Near-infrared fluorescent probes based on piperazine-functionalized BODIPY dyes for sensitive detection of lysosomal pH

Article abstract of DOI:10.1039/c4tb01878h

Three acidotropic, near-infrared fluorescent probes based on piperazine-modified BODIPY dyes (A, B and C) have been developed for the sensitive and selective detection of lysosomal pH in living cells. Probes A and B display low solubilities in aqueous solutions, whereas probe C is highly water-soluble. The fluorescent responsive mechanism of these probes to lysosomal pH is based on intramolecular charge transfer (ICT) and potential photo-induced electron transfer from piperazine moieties at 3,5-positions to BODIPY cores in the near-infrared region. The sensitivity and selectivity of the probes to pH over metal ions have been investigated by spectroscopic analysis in aqueous solutions. The probes have low auto-fluorescence at physiological pH conditions, whereas their fluorescence intensities significantly increase when pH is shifted to an acidic condition. Furthermore, these three probes were successfully applied to the in vitro lysosome imaging inside normal endothelial and breast cancer cells. This journal is

Full text of DOI:10.1039/c4tb01878h

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ARTICLE
Near-infrared Fluorescent Probes Based on
Piperazine-Functionalized BODIPY Dyes for
Sensitive Detection of Lysosomal pH
Cite this: DOI: 10.1039/x0xx00000x
Jingtuo Zhanga, Mu Yanga, Cong Lia, Nethaniah Dorha, Fei Xiea, Fen-Tair Luo*b,
Ashutosh Tiwari*a, and Haiying Liu*a
Received 00th January 2015,
Accepted 00th January 2015
DOI: 10.1039/x0xx00000x
Three acidotropic, near-infrared fluorescent probes based on piperazine-modified BODIPY dyes (
and ) have been developed for sensitive and selective detection of lysosomal pH in living cells. Probes
and display low solubilities in aqueous solutions while probe is highly water-soluble. The
A, B
www.rsc.org/
C
A
B
C
fluorescent responsive mechanism of these probes to lysosomal pH is based on intramolecular charge
transfer (ICT) and potential photoinduced electron transfer from piperazine moieties at 3,5-positions to
BODIPY cores in near-infrared region. The sensitivity and selectivity of the probes to pH over metal ions
have been investigated by spectroscopic analysis in aqueous solutions. The probes have low auto-
fluorescence at physiological pH condition while their fluorescence intensities significantly increase
when pH is shifted to acidic condition. Furthermore, these three probes were successfully applied to the
in vitro lysosome imaging inside normal endothelial and breast cancer cells.
The potential drawback for these fluorescent probes is their
broad pH responses and relative high fluorescent background at
pH 7.4. Recent approach to address this high fluorescent
background at pH 7.4 is to use fluorescein and Rhodamine dyes
by taking advantages of low fluorescence background at pH 7.4
1. Introduction
Lysosomes, membraneꢀdelimited, acidic organelles present in
essentially all eukaryotic cells, employ more than 70 hydrolytic
enzymes to degrade biological macromolecules taken up by
endocytosis, phagocytosis and autophagy.1ꢀ3 Significant
disruption in the lysosomal pH can cause lysosome malfunction
and thus cause lysosomal storage diseases.4 Therefore, it is very
important to precisely detect lysosomal pH in living cells in
order to study cellular functions for insightful understanding of
physiological and pathological processes. Fluorescence
spectroscopy employing pHꢀsensitive fluorescent probes is
becoming one of the most powerful tools for monitoring
intracellular pH, and possesses many technical and practical
advantages over other methods because it can detect the
intracellular pH of intact cells and subcellular regions with
operational simplicity, high sensitivity, and excellent spatial
and temporal resolution.5ꢀ15 Only a few of them have been
applied to detect lysosomal pH inside living cells even though
many fluorescent probes for pH have been prepared.16ꢀ21 Most
fluorescent probes are selectively accumulated in acidic
lysosomes via lysosomotropism where their tertiary amine
residues are protonated in a cellular acidic environment.12 The
protonation of ionizable tertiary amine groups on the
fluorophores in acidic lysosomes enhances the probe
fluorescence through the suppression of photoꢀinduced electron
transfer from the tertiary amine to the probe fluorophores.9, 22ꢀ26
because of the ‘‘ringꢀclosed’’ state of spirolactam rings in the
7, 14, 27
fluorophores at pH 7.4.6,
However, most fluorescent
probes are not soluble in aqueous solution, and some of them
can cause cell damage due to their short absorption and
emission wavelengths with less than 600 nm as very few nearꢀ
infrared fluorescent probes for lysosomal pH in living cells
were reported.28ꢀ30 In order to address these issues of high
fluorescent background and potential cell damage, we reported
Rhodamine dye counterparts with nearꢀinfrared excitation and
emission wavelengths as fluorescent probes for lysosomal pH
in living cells.31 We have demonstrated the feasibility of nearꢀ
infrared fluorescent probes with spirocyclic structures for
sensitive detection of lysosomal pH in living cells with
advantages of excellent photostability, low cytotoxicity, deep
tissue light penetration and low autofluorescence background.31
Under neutral or basic conditions, the fluorescent probes retain
the spirocyclic form that is nonꢀfluorescent and colorless.
Acidic environment effectively leads to ring opening of the
spirocylic form in fluorescent probes, and results in strong
fluorescence. However, these fluorescent probes are still
insoluble in aqueous solution.31
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DOI: 10.1039/C4TB01878H
The search for readily accessible nearꢀinfrared fluorescent
2. Experimental Section
probes with good water solubility, large dynamic range and
high specificity is still a challenging task for nearꢀinfrared
imaging of lysosomal pH in living cells. In this paper, we
design and prepare nearꢀinfrared fluorescent probes for
lysosomal pH by using piperazine residues to manipulate the
fluorescent responses of the probes to pH via modulation of
intramolecular charge transfer (ICT) effect and potential
photoinduced electron transfer of piperazine moieties at 3,5ꢀ
positions to BODIPY cores at different pH values. Piperazine
residues were incorporated into BODIPY dyes at 3,5ꢀpositions
as parts of BODIPY cores while tri(ethylene glycol)methyl
ether residues attached to piperazine moieties were used to
enhance hydrophilic properties of the probes and improve water
solubility of the probes. Fluorescent probes display very weak
fluorescence because of strong intramolecular charge transfer
from piperazine residues to BODIPY cores at neutral pH, and
potential photoꢀinduced electron transfer from lower part
nitrogen atoms of piperazine groups to BODIPY core.
Decrease of pH significantly enhances fluorescence intensity
because the pronation of piperazine residues at acidic pH
considerably reduces intramolecular charge transfer effect of
the piperazine residues to BODPY cores, and the potential
photoꢀinduced electron transfer effect. These fluorescent probes
have advantages such as high photostability, sensitive and
selective nearꢀinfrared imaging of lysosomal pH in living cells
with potential intact in vivo imaging and deep tissue penetration
without autoꢀfluorescence and unintended cellular damage
issues.
2.1 Instrumentations:
1H NMR and 13C NMR spectra were collected on 400 MHz
Varian Unity Inova NMR spectrophotometer instrument. 1H
and 13C NMR spectra were recorded in CDCl3 and DMSOꢀd6.
Chemical shifts (δ) were given in ppm relative to solvent
residual peaks (1H: δ 7.26 for CDCl3, δ 2.50 for DMSOꢀd6 13C:
;
δ 77.3 for CDCl3) as internal standard. HRMS were measured
with electrospray ionization (ESI) mass spectrometer and
electron impact ionization (EI) mass spectrometer. Absorption
spectra were taken on a Perkin Elmer Lambda 35 UV/VIS
spectrometer. Fluorescence spectra were recorded on a Jobin
Yvon Fluoromaxꢀ4 spectrofluorometer. The potential
aggregation behaviors of probes
solutions were determined by dynamic light scattering (Coulter
NP4 Plus, Beckman Coulter, Fullerton, CA).
A, B and C in aqueous
2.2 Materials:
Unless specifically indicated, all reagents and solvents were
obtained from commercial suppliers and used without further
purification. BODIPY dyes
7
and
9 were prepared from
compounds
6 and 8 respectively, according to our previous
reported procedures32, 33
General procedure to prepare fluorescent probe A, B
and C: When BODIPY dye
eq.) were dissolved in benzene, a small amount of piperidine
and acetic acid were added into the solution and the reaction
mixture was refluxed for 10ꢀ16 hours at 100 C. Any water
7 or 8 (1 eq.), compound 3 or 5 (6
o
formed during the reaction was removed azeotropically by
using a DeanꢀStark apparatus. After the reaction was quenched
by water at room temperature, the mixture was concentrated
under reduced pressure and reꢀdissolved in 100 mL
dichloromethane. It was sequentially washed with water (2 x
100 mL) and brine (100 mL), dried over Na2SO4 and filtered.
The filtrate was concentrated under reduced pressure and the
crude product was purified by column chromatography with
silica gel (32ꢀ63 microns, 230ꢀ400 mesh) by using
CH2Cl2/MeOH (7/1, v/v) as eluent to obtain fluorescent probes
A
,
B
or
Fluorescent probe A: According to the general procedure,
fluorescent probe was prepared from BODIPY dye (50 mg,
0.077 mmol), compound (95 mg, 0.465 mmol), benzene (8
C:
A
7
3
mL), piperidine (0.1 mL, 1.012 mmol) and acetic acid (0.1 mL,
1.747 mmol). The mixture was refluxed for 10 hours. After
workꢀup and purification by column chromatography by using
CH2Cl2/MeOH/Et3N (7/1/0.01, v/v/v) as eluent, probe
A
was
obtained as green solid (33 mg, 42%). H NMR (400 MHz,
CDCl3) 7.67 – 7.49 (m, 6H), 7.25 – 7.12 (m, 2H), 6.99 (d,
= 8 Hz, 1H), 6.95 – 6.87 (m, 5H), 6.84 (d, = 8 Hz, 1H), 6.60
(br s, 2H), 4.22 (t, = 4.8 Hz, 2H), 4.15 (t, = 4.8 Hz, 2H),
3.92 (t, = 4.8 Hz, 2H), 3.86 (t, = 4.8 Hz, 2H), 3.81 – 3.49
(m, 16H), 3.42 – 3.30 (m, 14H), 2.68 (br s, 8H), 2.43 (s, 6H),
1.51 (s, 6H); 13C NMR (100 MHz, CDCl3)
152.9, 151.4,
1
δ
J
J
J
J
J
J
Scheme 1 Chemical structures of near-infrared fluorescent probes and their
responses to pH.
δ
149.8, 149.7, 141.7, 136.1, 133.6, 129.2, 128.4, 128.3, 122.0,
2 | Journal of Materials Chemistry B, 00, 1-3
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DOI: 10.1039/C4TB01878H
117.6, 116.8, 115.7, 115.2, 114.7, 72.2, 72.2, 71.2, 71.0, 70.9, 2.4 Cell culture and fluorescence imaging
70.1, 70.0, 69.4, 69.0, 59.3, 54.9, 48.1, 46.0, 15.0. HRMS
Breast cancer cells MDAꢀMBꢀ231 and normal endothelial cells
(ESI): calculated for C57H75BF2N6O8 [M]+, 1020.5702; found,
1020.5736.
HUVECꢀC (from ATCC) were cultured as previously
described.34 In brief, cells were plated on 12ꢀwell culture plates
at 1 x 105 cells/well and incubated at 37 oC in 5% CO2
incubator overnight. Next day, the media was removed and
cells were rinsed twice with 1 X PBS after which fresh serumꢀ
Fluorescent probe B: According to the general procedure,
fluorescent probe
B
was prepared from BODIPY dye
7 (50 mg,
0.077 mmol), compound
5
(157 mg, 0.467 mmol), benzene (10
mL), piperidine (0.1 mL, 1.012 mmol) and acetic acid (0.1 mL,
1.747 mmol). The mixture was refluxed for 10 hours. After
workꢀup and purification by column chromatography by using
0
free media was added and cells were incubated for 2 h at 37 C
in CO2 incubator. Following 2 h serum starvation, fresh serum
free media with/without 2 µM, 5 µM, 10 µM probe
A or 5 µM,
CH2Cl2/MeOH (7/1, v/v) as eluent, probe
B
was obtained as
green oil (51 mg, 52%). H NMR (400 MHz, CDCl3) 7.60 –
7.47 (m, 6H), 7.16 (d, = 16 Hz, 2H), 6.97 (d, = 8 Hz, 1H),
6.91 – 6.85 (m, 5H), 6.81 (d, = 8 Hz, 1H), 6.59 (s, 2H), 4.20
(t, = 4.8 Hz, 2H), 4.12 (t, = 4.8 Hz, 2H), 3.89 (t, = 4.8 Hz,
2H), 3.84 (t, = 4.8 Hz, 2H), 3.79 – 3.47 (m, 36H), 3.38 –
3.36 (m, 9H), 3.32 (s, 3H), 3.30 (br s, 8H), 2.75 – 2.64 (m,
12H), 1.49 (s, 6H); 13C NMR (100 MHz, CDCl3)
152.8,
15 µM, 25 µM probe or were added and incubated further
B
C
1
δ
with cells for 2 h. Live cell images were acquired using an
inverted fluorescence microscope (AMFꢀ4306, EVOSfl, AMG)
with DAPI filter for Hoechst 33342 (SigmaꢀAldich), GFP filter
for LysoSensor Green DNDꢀ189 (Invitrogen), and CY5 filter
J
J
J
J
J
J
J
for fluorescent probes
A, B, and C. The fluorescence images
were obtained at 40x and 60x magnification for HUVECꢀC and
at 60x magnification for MDAꢀMBꢀ231 cells. The exposure
times for each filter were kept constant. Coꢀlocalization
analysis based on Pearson’s coefficient was done using JACoP
plugin from ImageJ.35
Live cell fluorescence imaging at different intracellular
pH: Normal endothelial cells HUVECꢀC (from ATCC) were
cultured as previously described. In brief, cells were plated on
12ꢀwell culture plates at 1 x 105 cells/well and incubated at 37
oC in 5% CO2 incubator overnight. Next day, when the media
was removed and cells were rinsed twice with 1 X PBS (pH
δ
151.6, 149.7, 149.6, 142.8, 141.5, 137.1, 136.1, 133.5, 130.1,
129.0, 128.4, 127.9, 125.6, 121.9, 117.5, 116.5, 115.4, 115.2,
115.0, 114.6, 72.2, 72.1, 71.1, 70.9, 70.8, 70.7, 70.6, 70.0, 69.9,
69.3, 69.1, 68.9, 59.3, 59.2, 48.3, 47.9, 14.9. HRMS (ESI):
calculated for C69H99BF2N6O14 [M]+, 1284.7274; found,
1284.7261.
Fluorescent probe C: According to the general procedure,
fluorescent probe
C
was prepared from BODIPY dye
9 (50 mg,
0.045 mmol), compound
5
(90 mg, 0.268 mmol), benzene (8
mL), piperidine (0.1 mL, 1.012 mmol) and acetic acid (0.1 mL,
1.747 mmol). The mixture was refluxed for 16 hours. After
workꢀup and purification by column chromatography by using
7.4), fresh serum free media with 5 µM probe
A or 15 µM
probe or 15 µM probe was added and incubated further
B
C
with cells for 2 h. After incubation, the medium was removed
and cells were gently rinsed with PBS (pH 7.4) three times. It
was then treated with nigericin (5 ꢁg/mL) in 2 mL potassium
rich PBS at different pH values of 5.0, 5.5, 6.5, and 7.5, and
incubated further for 15 min. Live cell images were acquired
using an inverted fluorescence microscope (AMFꢀ4306,
EVOSfl, AMG) with DAPI filter for Hoechst 33342 (Sigmaꢀ
Aldich), GFP filter for LysoSensor Green DNDꢀ189
CH2Cl2/MeOH (7/1, v/v) as eluent, probe
C
was obtained as
green oil (30 mg, 38%); H NMR (400 MHz, DMSOꢀd6
7.50 – 7.41 (m, 6H), 7.35 (d, = 16 Hz, 2H), 7.13 (d,
Hz, 1H), 7.04 – 6.97 (m, 5H), 6.91 – 6.87 (m, 3H), 4.06 (d,
8 Hz, 2H), 4.01 (d, = 8 Hz, 2H), 3.62 – 3.36 (m, 76H), 3.34 –
3.18 (m, 26H), 2.59 (br s, 12H), 2.31 (sextet, = 5.6 Hz, 1H),
2.25 (sextet,
= 5.6 Hz, 1H) 1.50 (s, 6H); 13C NMR (100 MHz,
CDCl3) 152.9, 151.5, 149.9, 149.8, 142.7, 141.7, 137.6,
1
)
δ
J
J = 8
J
=
J
J
J
δ
(Invitrogen), and CY5 filter for fluorescent probes
A, B, and C.
136.0, 133.6, 130.1, 129.1, 128.3, 127.9, 121.5, 117.5, 116.7,
115.6, 114.3, 113.9, 72.2, 72.2, 71.1, 70.9, 70.9, 70.8, 70.7,
70.7, 69.7, 69.6, 67.8, 67.4, 59.3, 59.2, 57.9, 48.1, 32.2, 23.0,
15.1, 14.4. HRMS (ESI): calculated for C91H143BF2N6O24 [M]+, 2.5 Determination of cellular uptake efficiency
The fluorescence images were obtained at 40x magnification.
The exposure times for each filter were kept constant.
1753.0209; found, 1753.0194.
HUVECꢀC cells were plated at a density of 1 × 104 cells/well
on a 96ꢀwell cell culture plate and incubated at 37 C in 5%
CO2 incubator overnight. Next day, the culture medium was
removed and cells were rinsed twice with PBS (pH 7.4). Fresh
o
2.3 Optical measurements
All absorption and emission spectra were recorded by using
standard 1 cm path length quartz fluorescence cuvette at room
temperature. The slit widths of excitation and emission were set
to 3 nm and 5 nm respectively, and the excitation wavelength is
set at 620 nm for fluorescence spectroscopies. 1.0 mM DMSO
100 µL media with 2 µM, 5 µM, 10 µM of probe
A, or 5 µM,
15 µM, 25 µM of probe or 5 µM, 15 µM, 25 µM of probe
B
C
were added to the wells in triplicate and incubated for 2 h.
Controls and blanks were also set in triplicate at the same time.
Controls had culture media and dyes but no cells. Blanks used
for background subtraction had cells and media but no dyes.
After 2 h incubation the media (100 µL) was pipetted out from
the plate and put in a fresh 96ꢀwell plate. Each well was rinsed
with 100 µL of fresh PBS (pH 7.4) and the solution was added
solutions of fluorescent probes
solution of probe were used as stock solutions. All optical
spectra of fluorescent probes and were measured in the
50 mM citrateꢀphosphate buffer solutions (contain 0.5% DMSO
from stocks for fluorescent probes and ).
A and B and 1.0 mM aqueous
C
A
,
B
C
A
B
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DOI: 10.1039/C4TB01878H
to the respective wells in the fresh 96ꢀwell plate. Then In order to conjugate piperazine moiety into BODIPY dyes as
absorbance of wells was measured using an ELISA plate reader parts of the fluorophores to modulate intramolecular charge
(BioTek Instruments, Inc.) at 712 nm, 710 nm, and 687 nm transfer from the piperazine moieties to BODIPY cores
(absorbance peak of fluorescent probes
respectively). Values of respective blanks were subtracted from yl)benzaldehyde
samples (Adye) and controls (Actrl). Cellular uptake efficiency methoxyethoxy)ethoxy)ethyl)piperazinꢀ1ꢀyl)benzaldehyde
A
,
B
,
and
C
,
(Scheme
2),
we
prepared
(3)
4ꢀ(4ꢀmethylpiperazinꢀ1ꢀ
and 4ꢀ(4ꢀ(2ꢀ(2ꢀ(2ꢀ
(
5
4
)
)
was calculated as [1ꢀ (Adye / Actrl)]×100%.
by reacting 4ꢀfluorobenzaldehyde (
1
) with 1ꢀmethylpiperazine
(
2
) and 1ꢀ(2ꢀ(2ꢀ(2ꢀmethoxyethoxy)ethoxy)ethyl)piperazine (
2.6 MTS assay
o
in DMF solution under a basic condition at 80 C, respectively.
was prepared
MTS assay was performed with MDAꢀMBꢀ231 cells (ATCC) BODIPYꢀbased nearꢀinfrared fluorescent probe
A
as previously described.36 Briefly, the cells were plated at a by incorporating piperazine residues into BODIPY cores at
density of 5000 cells/well on a 96ꢀwell cell culture plate and 3,5ꢀpositions via the Knoevenagel condensation reaction of our
incubated at 37 oC in 5% CO2 incubator overnight. After previously reported BODIPY dye
(
7) with 4ꢀ(4ꢀ
). Fluorescent probe is
with 1 X PBS. Fresh media with 0 µM, 5 µM, 15 µM, 25 µM, hydrophobic and displays very low solubility with less than 0.1
and 50 µM of fluorescent probes , and dissolved in mg/mL in water. In order to enhance hydrophilic feature of the
DMSO (with less than <0.5% DMSO final concentration in fluorescent probes, we prepared nearꢀinfrared emissive probes
media) were added to the wells and measured in 6 replicates for and by using hydrophilic 4ꢀ(4ꢀ(2ꢀ(2ꢀ(2ꢀ
each dye concentration, respectively. Blanks that had methoxyethoxy)ethoxy)ethyl)piperazinꢀ1ꢀyl)benzaldehyde
everything else except the cells were prepared at the same time. to conduct the Knoevenagel condensation reactions with
incubation, the media was removed and the cells were washed methylpiperazinꢀ1ꢀyl)benzaldehyde (
3
A
A
,
B
C
(
B
C)
(5)
o
The plates were incubated at 37 C in 5% CO2 incubator for 72 BODIPY dye (
h. After the 72ꢀhour incubation, 20 µL of MTS solution (from BODIPY dye (
CellTiter 96 Aqueous NonꢀRadioactive Cell proliferation Assay mg/mL) than probe
(MTS) kit, Promega) was added to each well. The absorbance solubility with at least 20 mg/mL in aqueous solution. The
7
) and our previously reported water soluble
respectively. Probe is more soluble (~1.0
in water while probe shows excellent
9
)
,
B
A
C
o
at 490 nm was acquired after 4ꢀhour incubation at 37 C, using hydrophilicity of fluorescent probes
B and C are significantly
an ELISA plate reader (BioTek Instruments, Inc.) Plots were enhanced because of the numerous hydrophilic oligo(ethylene
normalized to control wells containing media and cells only.
glycol)methyl ether residues at mesoꢀ and 3,5ꢀpositions which
significantly facilitate the strong enthalpic interactions with
water molecules.
3. Results and Discussion
3.1 Synthesis approach
Scheme 2 Synthetic route to near-infrared fluorescent probes
4 | Journal of Materials Chemistry B, 00, 1-3
A, B and C.
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moieties at lower pH. Further decrease of pH to 2.6
significantly reduces the aggregation size of probe to 22 nm
) in ethanol is while probes and show no detectable aggregations in this
3.2 Optical properties of fluorescent probes
A
The absorption properties of BODIPY dye (
7
B
C
characterized by a strong S0
and a weaker broad band around 350 nm attributed to the
S0 S2 ( *) transition. It shows fluorescent peak at 509 nm
in ethanol solution. Fluorescent probe displays significant
red shifts in absorption and emission spectra in ethanol solution
with absorption and emission peaks at 668 nm and 715 nm,
respectively because of significantly enhanced πꢀconjugation
and intramolecular charge transfer effect from piperazine
moieties at 3,5ꢀpositions to the BODIPY core compared with
ꢀS1 (πꢀπ*) transition at 499 nm condition. (Fig. S37ꢀS40 in ESI†).
ꢀ
πꢀπ
Table 1 Photophysical properties of probes A, B and C (protonation and
nonꢀprotonation form). The protonation source in organic solvent is
trifluoroacetic acid.
A
λabs
(nm)
λem
εmax
Φf
solvent
(nm)
(104 M-1cm-1)
(%)
7
9
Ethanol
Ethanol
499
500
675
668
674
712
658
652
675
680
679
670
676
710
662
653
669
675
675
669
675
687
661
653
665
671
509
509
722
715
721
770
690
685
715
716
729
720
724
755
696
683
715
715
722
720
724
750
694
687
715
716
9.10
7.14
7.10
6.88
7.05
4.36
7.58
7.86
4.35
3.94
9.31
8.56
9.27
5.20
10.4
11.2
5.60
4.60
7.76
7.88
8.11
4.80
8.55
9.22
5.92
4.46
72.1
61.0
4.3
BODIPY dye (
probe were obtained in dichloromethane and DMSO
solutions. However, in aqueous solution the intramolecular
charge transfer effect of the probe becomes more significant
7). The similar spectroscopic properties of
CH2Cl2
A
Ethanol
3.8
A
A+H+
B
DMSO
3.4
A
because of polar solvent effect and potential dye aggregation
may occur, which results in very low fluorescence and further
Buffer (pH 7.4)
CH2Cl2
<0.01
11.5
10.3
0.033
0.01
3.4
red shifts in absorption and emission spectra of probe
absorption and emission peaks at 712 nm and 770 nm in buffer
(pH 7.4), respectively (Table 1). Fluorescent probe exhibits
similar optical properties to those of the probe in organic
solvents. But the absorption peak (710 nm) and emission peak
(755 nm) of probe in buffer solution (pH 7.4) were found
slightly blue shifted compared with probe (Table 1). This
A with
Ethanol
Buffer (pH 2.6)
Buffer (pH 4.5)
CH2Cl2
B
A
Ethanol
2.8
B
DMSO
3.0
A
Buffer (pH 7.4)
CH2Cl2
<0.01
9.2
may be attributed to the reduced aggregation in aqueous
solution with hydrophilic oligo(ethylene glycol)methyl ether
residues on piperazine moieties at 3,5ꢀpositions. Highly waterꢀ
Ethanol
10.3
0.06
0.02
4.5
B+H+
soluble BODIPY dye (
peaks at 500 nm and 509 nm in aqueous solution. Fluorescent
probe shows considerable red shifts in absorption and
9) displays absorption and emission
Buffer (pH 2.6)
Buffer (pH 4.5)
CH2Cl2
C
emission spectra with absorption peaks at 669 nm and 687 nm,
and emission peaks at 720 nm and 750 nm in ethanol and buffer
solution (pH 7.4), respectively (Table 1) because of its
significant extended πꢀconjugation and intramolecular charge
transfer effect from piperazine moieties at 3,5ꢀpositions to the
BODIPY core compared with those of its precursor BODIPY
Ethanol
2.8
C
DMSO
3.3
Buffer (pH 7.4)
CH2Cl2
<0.01
9.9
Ethanol
11.0
0.16
0.07
C+H+
Buffer (pH 2.6)
Buffer (pH 4.5)
dye
9
. Compared with the properties of probes
A and B, the
highly waterꢀsoluble character of probe
C
may further reduce
its selfꢀaggregation effect in buffer solution as it shows the
shortest absorption and emission wavelength among three
probes in buffer solution (pH 7.4). Light scattering
measurements confirmed the presence of aggregated species of
3.3 Optical responses of fluorescent probes to pH
We investigated pH effect on optical properties of fluorescent
probes and their BODIPY precursors ( ) in buffer solutions.
We did this to verify our hypothesis that incorporation of
piperazine moieties into BODIPY dyes at 3,5ꢀpositions can
effectively modulate sensitive optical responses of BODIPY
probes
A, B and C in aqueous solution at pH 7.4 with average
7, 9
aggregation sizes (hydrodynamic diameter) of 500 nm, 334 nm
and 272 nm, respectively. Decrease of pH to 4.5 reduces the
aggregation sizes of fluorescent probes
A, B and C to 391 nm,
174 nm and 82 nm respectively because of charge repulsion
between BODIY dyes through protonation of piperazine
dyes to pH. BODIPY dyes
7 and 9 display no responses to pH
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in absorption and emission in buffer solution, which are typical piperazine moieties as well as reduced aggregation effect (Fig.
optical properties of typical BODIPY dyes (Fig. S20ꢀS22 in S37ꢀS40 in ESI†) in acidic conditions (Scheme 1), which is
electronic supplemental information ESI†).
fluorescent probes and display very sensitive responses and 23 nm in absorption spectra of fluorescent probes
to pH in both absorption and emission (Fig. 1). Decreases of pH when pH values of the buffer were changed from 9.98 to
values from 9.98 to 2.20 cause significant increases in 2.20. Except ICT effect, fluorescence quenching caused by
fluorescent intensities at 715 nm of fluorescent probes and potential photoꢀinduced electron transfer (PET) from lower
with 75ꢀ, 88ꢀ and 102ꢀfold enhancements, respectively (Fig. nitrogen atoms of piperazine moieties at 3,5ꢀpositions of
1). In addition, there are significant blue shifts of 55 nm, 41 nm BODIPY core may also contribute to the extreme low
and 35 nm in emission spectra of the probes and fluorescence of all three probes in neutral condition. This
However, further confirmed by significant blue shifts of 46 nm, 53 nm
A
,
B
C
A, B
and
C
A, B
C
A
,
B
C,
respectively, because of significantly reduced intramolecular fluorescence quenching effect could be reduced at low pH
charge transfer (ICT) effect from piperazine moieties to because the protonation of piperazine moieties can prohibit the
BODIPY cores through protonation of nitrogen atoms in PET process.
Fig. 1 Emission (upper row) and absorption (lower row) spectra of fluorescent probes A, B and C (5 μM) in buffer solution at different pH values.
Fig. 2 shows more detailed fluorescent intensity change at 4.2, very good linear fluorescence responses were found for all
715 nm of probes and at different pH conditions in probes in which probe gives the highest sensitivity (Fig. 2
buffer solution. From basic to neutral condition, all probes inset). From pH 7.4 to pH 4.2, the fluorescence intensities
show very weak fluorescence without any obvious change. increased by 7ꢀ, 17ꢀ and 22ꢀfolds for probes and C,
Their fluorescence intensities gradually increase when pH respectively, and their fluorescence quantum yields at pH 4.5
decreases from 7.0 to 2.2. The pKa values of probes and also significantly increase compared with those in neutral pH
A
,
B
C
C
A, B
A
,
B
C
are 2.91, 3.19 and 3.57 respectively (see supplementary condition at 7.4 (Table 1). These linear, high dynamic range
information ESI† for method of pKa calculations). Obviously, and sensitive responses to pH indicate the feasibility of
probe
Ka value among all three probes. This indicates that the highly decreases of pH values from 2.20 to 1.5 results in significant
waterꢀsoluble probe has less aggregation and it is protonated fluorescence quenching of the probes (Fig. 2). We have
much more easily in aqueous solution. As a result, probe eliminated the possibility of decomposition of the probes as the
displays more sensitivity to pH than probes and . In the pH probes display stable reversible responses to pH between 1.5
C exhibits highest fluorescence responses with highest intracellular pH (pHi) detection by using these probes. Further
p
C
C
A
B
window between physiological pH at 7.4 and lysosomal pH at
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and 10. However, the fluorescence quenching mechanism of the spectra of the probe immediately changed. All peaks turned to
probes at extremely low pH needs further investigation. be very sharp shape and protons and significantly shifted to
j
k
In addition, we investigated the optical properties of the downꢀfiled (from 2.53 to 3.38 ppm) which indicates the
probes after protonation in organic solvents. After adding two protonation of the lower nitrogen atoms on piperazine moieties
equivalents of trifluoroacetic acid, the absorption and emission at 3,5ꢀpositions as it has more loneꢀpair availability to be
spectra of probe
dichloromethane solution. The dramatic blue shifts both on conjugated to BODIPY core. The neighbouring proton
absorption and emission were present with new absorption and downꢀfield shifted after protonation but with smaller change of
emission peaks at 652 nm and 685 nm in ethanol, and 658 nm 0.35 ppm (3.14 to 3.49 ppm). In addition, the aromatic protons
and 690 nm in dichloromethane solution, respectively. The and also underwent downꢀfield shifts after protonation and
fluorescence quantum yield of probe is also enhanced by these protons did not further shift with addition of more than
A
immediately changed in ethanol and protonated than the upper nitrogen atoms which were
i
also
f
b
A
2.71ꢀfold in ethanol and 2.67ꢀfold in dichloromethane solution. two equivalents of the acid. These results further confirm our
The disappearance of original peaks and presence of new blue previous hypothesis that the proton is transferred or delocalized
shifted peaks indicate that probe
A was fully protonated and the between the two nitrogen atoms of the piperazine moiety after
intramolecular charge transfer from nitrogen atoms at 3,5ꢀ protonation of the probes, which significantly reduces the
positions to BODIPY core was prohibited which enhanced the electron donation ability of conjugated nitrogen atoms to the
fluorescence. Probes
B and C exhibit the similar change after BODIPY core. Due to the protonation of the lower nitrogen
protonation in ethanol and dichloromethane. Surprisingly, there atoms at 3,5ꢀpositions, photoꢀinduced electron transfer from
is no change after adding trifluoroacetic acid in DMSO solution lower nitrogen atoms to BODIPY core may be prohibited
of probes. It may be due to the relative high pKa value of which can also result in the great increase of fluorescent
trifluoroacetic acid in DMSO (pKa = 3.45).37
intensity at lower pH (Fig. 1).38
3.5 Solvent effect
8.0
Considering the nature of the intramolecular charge transfer
(ICT) and potential selfꢀaggregations on BODIPY dyes, we
investigated the solvent polarity dependency of fluorescent
2
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
probe A
probe B
probe C
1
0
probes
A, B and C (Fig. 4). Optical properties of probes are
highly dependent on solvent polarity and they exhibit the same
changing patterns in their absorption and emission spectra in
different polarity environments. Decreases of the solvent
polarity by raising the percentage of ethanol in buffer solutions
result in slight blue shifts of the fluorescent peaks, and
significant fluorescence intensity enhancements of the probes.
These changes may result from the reduced selfꢀaggregation
effect via fluorescence selfꢀquenching, and more importantly
from reduced intramolecular charge transfer (ICT) excited
states of the fluorophores by nonpolar character of alcohol
compared with water which recovers fluorescence given by
more dominant locally excited (LE) states of the probes.
4
5
6
7
pH
pkaprobe A = 2.91
pkaprobe B = 3.19
pkaprobe C = 3.57
0
2
4
6
8
10
pH
Fig. 2 Fluorescent responses of probes
buffer solution. Inset: zoomed fluorescence intensity responses of probes
and to pH in the window between cytoplasm pH to lysosomal pH.
A, B and C to different pH values in
A, B
C
Similar blue shifts in absorption spectra of the probes
were observed with increasing percentage of alcohol in the
mixed buffer. However, the probes and show more blue
shifts (44 nm and 40 nm) in their absorption peaks in pure
ethanol solution than probe which only gives an 18ꢀnm shift.
The more blue shifts in their absorption spectra in pure ethanol
solution indicate that the probes and undergo potential
aggregation in aqueous solution while the probe has much
A, B and
C
A
B
3.4 NMR study
In order to further study intramolecular charge transfer effect of
piperazine moieties to BODIPY cores, we chose probe and
use 1H NMR to investigate the structure change before and after
protonation of the probe. Deuterium methanol (CD3OD) was
used as solvent for 1H NMR measurement because it gave
spectra with less peak overlaps than the spectra obtained in
CDCl3 solution. Fig. 3 shows the comparison of 1H NMR
B before and after its protonation in CD3OD
solution. Broad peaks were observed on the spectra of probe
due to the fast relaxation from its high polarity character and
potential hydrogen bonding on the nitrogen atom of piperazine
moiety. After adding two equivalents of trifluoroacetic acid, the
C
B
A
B
C
weaker aggregation effect because of its highly waterꢀsoluble
character as the bulky and hydrophilic branched oligo(ethylene
glycol)methyl ether residues on the fluorophore could
effectively prevent aggregation between BODIPY cores in
aqueous solution.
spectra of probe
B
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Fig. 3 1H NMR spectra (partial, peak
a – m) of probe B before and after addition of two equivalents of trifluoroacetic acid in CD3OD solution.
Fig. 4 Emission (upper row) and absorption (lower row) spectra of fluorescent probes
percentages of ethanol.
A
,
B
and
C
(5 μM) in mixed solvent (ethanol/buffer pH 7.4) with different
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Fig. 5 Fluorescence responses of fluorescent probes
A
,
B
and
C
(5 μM) to different cations in pH 4.5 and 7.4 buffer solution.
3.7 Photostability
3.6 Selectivity
The photostability measurement was carried in buffer solution
at pH 4.5. Fluorescent probes were excited continuously at 620
nm for 5 hours and fluorescence intensity was measured every
5 minutes. The result clearly showed that there was no
significant decrease of the fluorescence intensity under 5ꢀhour
excitation for all three probes (Fig. 6). The fluorescence
We studied effect of different metal ions on fluorescent
responses of fluorescent probes to pH and investigated potential
coordination of fluorescent probes with heavy, transition, and
main group metal ions. The fluorescent probes display high
selectivity to pH over these alkali, alkalineꢀearth metal ions,
and transitional metal ions because fluorescent probes
A
,
B
and
intensities of probes
A and B increased by 20% and 7% in first
C
display no responses to 200 µM alkali and alkalineꢀearth
80 and 30 minutes respectively, and keep almost constant level
after that. These increases may arise from the slow protonation
metal ions as such Na+, Mg2+ and Ca2+, as well as some
transitional metal ions (200 µM) such as Zn2+, Cu2+, Co2+, Ni2+,
Mn2+ and Fe2+, at both pH 7.4 and 4.5 (Fig. 5).
of probes
aqueous solution. Probe
A
and
B
because of their relative low solubility in
exhibited very stable fluorescent
C
In addition, we investigated the potential effect of biological
thiol such as cysteine on our probes. The results showed that
there are negligible changes of the absorption and fluorescence
emission in first 2 hours under excitation and only 2% of
fluorescence intensity decreased after 5ꢀhour excitation.
spectra of 5 ꢁM probes
A, B and C in buffer solution after
3.8 Fluorescence responses in living cells
addition of 20 ꢁM cysteine to the buffer solution at pH 7.4 and
4.5 within 30 minutes (Fig. S24–S25 in ESI†). This indicates
that our probes are stable in presence of cysteine and the
responses of the probes to pH are not interfered by cysteine.
To find out if probes
A, B, and C could selectively stain
lysosomes or acidic organelles in cells, all three probes were
compared with commercial probe LysoSensor Green DNDꢀ189,
a known lysosome probe that specifically fluorescently stains
acidic organelles in cells. Live cell imaging in various
concentrations of probes
cell lines: breast cancer cell line MDAꢀMBꢀ231 and normal
endothelial cell line HUVECꢀC (Fig. 7 and 8). In both cell
A, B, and C were conducted using two
3.0
Probe A
Probe B
Probe C
2.5
2.0
1.5
1.0
0.5
types, probes
and probe
area in the cell stained with probe
A
and
showed weak fluorescence signal inside cells. The
or matched those
B showed very strong fluorescence signals
C
A
,
B
C
stained with LysoSensor Green DNDꢀ189. This suggests that
these new probes are able to target lysosomes or other acidic
cellular compartments in cells in
LysoSensor Green DNDꢀ189 and distinguish between different
regions inside cells based on pH. Coꢀlocalization analysis based
on the Pearson’s coefficient showed value of 0.85 or higher for
a manner similar to
0
50
100
150
Time (min)
200
250
300
probes
A, B, and C with LysoSensor Green DNDꢀ189
indicating presence of both probes in the same cellular
compartment (Fig. S34 in ESI†). The enlarged 60x
magnification images from both cell lines (Fig. S32ꢀ33 in ESI†)
Fig. 6 Fluorescence intensities at 715 nm of probes A, B and C (5 μM) as function
of time in 5 hours under excitation (620 nm) in citrate-phosphate buffer (pH 4.5).
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show high fluorescent area localized in the perinuclear region, most hydrophilic compound among the three probes, showed
which is in line with a previous report that shows lysosomes poor fluorescent signal even at high concentration of 25 µM
localized in the perinuclear region in clusters after serum (Fig. S28, S31 in ESI†). One of the possible reasons for this
40
starvation.39,
Probe
A
showed the highest fluorescent difference could be that probes
A
and
, so they could easier bind
at 2 µM concentration (Fig. S26, S29 in ESI†). The fluorescent or interact with lipophilic structures like micelles, lipososomes,
signals from probe was not as strong as probe , but it membranes in lysosomes which further activate the
showed measurable signal at 5 µM concentration and at higher fluorescence by depressing ICT effect of the fluorophores.41
concentrations (Fig. S27, S30 in ESI†). However, probe , the
B are more hydrophobic
intensity among all three probes and displayed good signal even and less steric hindered than probe
C
B
A
C
Fig. 7 Fluorescence images of MDA-MB-231 cells incubated with fluorescent probes A, B, and C. Cells incubated with 5 µM of respective probes for 2 h, post serum
starvation (2 h) and imaged for co-localization with 1 µM LysoSensor Green and (1 μg/mL) Hoechst 33342 stains. All images were acquired at 60x magnification using
inverted fluorescence microscope (AMF-4306, EVOSfl, AMG).
Fig. 8 Fluorescence images of HUVEC-C cells incubated with fluorescent probes A, B, and C. HUVEC-C cells were incubated with 5 µM of respective probes for 2 h,
post serum starvation (2 h) and imaged for co-localization with 1 µM LysoSensor Green and (1 μg/mL) Hoechst 33342 stains. Images were acquired using the using
inverted fluorescence microscope (AMF-4306, EVOSfl, AMG) at 40X magnification.
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efficiency with 10%, 3% and 3% for incubation concentration
of 5 ꢁM, 15 ꢁM and 25 ꢁM, respectively. This low cellular
60
50
40
30
20
10
0
Probe A
Probe B
Probe C
10 ꢀM
uptake of probe
weak signals of the fluorescence images in living cells
compared with probes and
C could be the main reason for its relatively
5 ꢀM
A
B.
2 ꢀM
In order to examine the pH dependency of the probes inside
the living cells, we conducted live cell fluorescence imaging at
different intracellular pH values. In this study, HUVECꢀC cells
were incubated with 5 ꢁM probe
A, 15 ꢁM probe B or 15 ꢁM
probe
C
, respectively, and then with nigericin (5 ꢁg mLꢀ1) in
5 ꢀM
5 ꢀM
buffer solution at different pH values from 5.0, 5.5, 6.0 to 7.5 to
equilibrate the intracellular and extracellular pH. This is a
standard approach that has been widely used to calibrate the
15 ꢀM
15 ꢀM
25 ꢀM
25 ꢀM
intracellular pH (pHi).41ꢀ45 The probe
C displayed very weak
fluorescence in cells near physiological pH (pH 7.5) while its
fluorescence intensity was significantly enhanced as pH
decreased from 7.5 to 5.0 (Fig. 10). This turnꢀon response of
Fig. 9 The HUVEC-C cells cellular uptake efficiency of probes
A, B and C at
different concentrations after two hour incubations. The cellular uptake
efficiency was calculated as the percentage of probe taken up by cells out of
total amount of probe in initial incubation solution. Cell density = 1 × 104
cells/well.
probe
C
to acidic pHi is in line with the trend of its pH response
is sensitive to
in buffer solution, which indicates that probe
C
pH not only in buffer solution, but also inside the living cells
because of reduced ICT effect and potential photoꢀinduced
electron transfer effect from piperazine moieties to BODIPY
It should be noted that the intensity of fluorescence in living
cells is also dependent on the cellular uptake. Therefore, we
further investigated the cellular uptake of HUVECꢀC cells with
core through protonation. Compared with probe
probe LysoSensor Green did not display any obvious change of
fluorescence at different pHi values. Probes and exhibited
similar response to different pHi as probe , but with smaller
C, commercial
different incubation concentrations of probes
A, B and C (Fig.
A
B
9). The cellular uptake efficiency was calculated as the
percentage of probe taken up by cells out of total amount of
C
fluorescence enhancements when pHi decreased from 7.5 to 5.0
(Fig. S35ꢀS36). This difference may be due to the fact that
probe in initial incubation solution. The results showed probe
has much higher cellular uptake (more than 30%) at all three
incubation concentrations compared with probes and
Probe has moderate cellular uptake efficiency of 18% at
A
probe
C
has higher pKa value and better water solubility than
and
B
C.
probes
A
B.
B
incubation concentration of 5 ꢁM but higher concentrations of
15 ꢁM and 25 ꢁM only give 8% and 4% uptake efficiency,
respectively. Probe
C showed the lowest cellular uptake
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VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C4TB01878H
Fig. 10 Fluorescence images of HUVEC-C cells incubated with 15 µM probes
as co-stains. Images were acquired using the using inverted fluorescence microscope (AMF-4306, EVOSfl, AMG) at 40X magnification.
C
at different pH values. 1 µM LysoSensor Green and 1 μg/mL Hoechst 33342 were used
University faculty startꢀup fund, and Research Excellence Fund
(to A. Tiwari).
1.2
1.0
0.8
0.6
0.4
0.2
0.0
5 ꢀM
15 ꢀM
25 ꢀM
50 ꢀM
Control
0.5% DMSO
Notes and references
a
Department of Chemistry, Michigan Technological University,
Houghton, MI 49931
b
Institute of Chemistry, Academia Sinica, Taipei, Taiwan 11529,
Republic of China
† Electronic Supplementary Information (ESI) available: [The details of
spectroscopic analysis, synthesis procedures of compound
spectra, absorption and emission spectra of probe and
solvents, the spectroscopic responses of BODIPY dye and
pH and fluorescence images of both cell lines with probe
different concentrations are included.]. See DOI: 10.1039/b000000x/
3 and 5, NMR
A
,
B
C
9
in different
to different
7
A, B and C at
Probe A
Probe B
Probe C
and C tested by 1. A. C. Johansson, H. Appelqvist, C. Nilsson, K. Kagedal, K. Roberg
Control
Fig. 11 Cytotoxicity and cell proliferation effect of probes
A
,
B
MTS assay. The MDA-MB-231 cells were incubated with 5, 15, 25, and 50 μM of
probes and for 72 h and cell viability was measured by adding MTS
reagent and measuring the formation of formazon at 490 nm. Cell viability is
and K. Ollinger, Apoptosis, 2010, 15, 527ꢀ540.
A
,
B
C
2. B. Turk and V. Turk, J. Biol. Chem., 2009, 284, 21783ꢀ21787.
directly proportional to the absorbance measured at 490 nm and was normalized 3. J. Stinchcombe, G. Bossi and G. M. Griffiths, Science, 2004, 305
,
to control cells (no probe added). The error bars indicate ± S.D. from 6 replicates.
55ꢀ59.
4. E. J. Blott and G. M. Griffiths, Nat. Rev. Mol. Cell Biol., 2002,
3
,
MTS assay were performed to investigate the toxicity of
these probes to cells (Fig. 11). Incubating the cells with the
122ꢀ131.
5. Z. J. Diwu, C. S. Chen, C. L. Zhang, D. H. Klaubert and R. P.
Haugland, Chem. Biol., 1999, , 411ꢀ418.
probes for 72 h shows that probe
A has the highest cytotoxicity
6
among the three probes with ~50% viability observed for cells
incubated with 5 µM that dropped down to less than 10%
viability at 15 µM and higher probe concentration (Fig. 11). In
6. H. S. Lv, J. Liu, J. Zhao, B. X. Zhao and J. Y. Miao, Sens. Actuator
B-Chem., 2013, 177, 956ꢀ963.
7. H. Zhu, J. L. Fan, Q. L. Xu, H. L. Li, J. Y. Wang, P. Gao and X. J.
Peng, Chem. Commun., 2012, 48, 11766ꢀ11768.
comparison, probes
B and C showed very mild toxicity with
greater than 70% cell viability even at 50 µM concentration
(Fig. 11).
8. Z. Li, Y. L. Song, Y. H. Yang, L. Yang, X. H. Huang, J. H. Han
and S. F. Han, Chem. Sci., 2012, 3, 2941ꢀ2948.
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10. D. G. Smith, B. K. McMahon, R. Pal and D. Parker, Chem.
Commun., 2012, 48, 8520ꢀ8522.
We have prepared three BODIPYꢀbased nearꢀinfrared
fluorescent probes
A, B and C for lysosomal pH. The response
11. L. J. Ma, W. G. Cao, J. L. Liu, D. Y. Deng, Y. Q. Wu, Y. H. Yan
and L. T. Yang, Sens. Actuator B-Chem., 2012, 169, 243ꢀ
247.
mechanism of the fluorescent probes to pH value relies on
modulation of intramolecular charge transfer and potential
photoꢀinduced electron transfer of piperazine moieties at 3,5ꢀ
positions to BODIPY cores. These probes displays extremely
low fluorescent at neutral pH because of intramolecular charge
transfer and potential photoꢀinduced electron transfer from
piperazine moieties to BODIPY cores, and become highly
fluorescent at low pH due to the significantly reduced
intramolecular charge transfer effect and potential photoꢀ
induced electron transfer effect. These fluorescent probes are
cellꢀpermeable and provide selective and sensitive labelling of
lysosomes for potential nonꢀinvasive monitoring of lysosomal
pH changes during physiological and pathological processes.
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Acknowledgments
This work was partially supported by National Science
Foundation (to H.Y. Liu), and Michigan Technological
Hu, G. H. Cheng and X. J. Peng, Dyes Pigment., 2013, 99,
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Journal of Materials Chemistry B, 00, 1-3 | 13

Journal of Materials Chemistry B
Page 14 of 14
View Article Online
DOI: 10.1039/C4TB01878H
Three BODIPY-based near-infrared fluorescent probes have been synthesized, characterized, and
evaluated for sensing and imaging of lysosomal pH inside living cells.