A porphyrin-pyranine dyad for ratiometric fluorescent sensing of intracellular pH

Article abstract of DOI:10.1016/j.jphotochem.2020.112524

The detection of intracellular pH (pH_i) by fluorescence imaging is highly desired for cell biology and medical science. In this study, we developed a FRET-based porphyrin-pyranine dyad (Por-Py 1) composed of a pyranine moiety as the energy dono

Full text of DOI:10.1016/j.jphotochem.2020.112524

Journal Pre-proof
A Porphyrin-pyranine Dyad for Ratiometric Fluorescent Sensing of
Intracellular pH
Jinghui Zhang (Investigation) (Conceptualization) (Writing - original
draft), Mengliang Zhu (Conceptualization) (Formal analysis), Jiawen
Cui, Chiming Wang (Investigation) (Data curation), Ziwen Zhou
(Investigation) (Data curation), Tianhang Wang (Investigation) (Data
curation), Lei Gong (Investigation) (Data curation), Chaorui Su,
Dongdong Qi (Investigation) (Data curation), Yongzhong Bian
(Conceptualization) (Supervision) (Validation) (Writing - review and
editing), Hongwu Du (Investigation)<ce:contributor-role>Resource),
Jianzhuang Jiang (Funding acquisition) (Project administration)
(Validation) (Writing - review and editing)
PII:
S1010-6030(20)30323-3
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112524
JPC 112524
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
10 February 2020
25 March 2020
26 March 2020
Please cite this article as: Zhang J, Zhu M, Cui J, Wang C, Zhou Z, Wang T, Gong L, Su C, Qi
D, Bian Y, Du H, Jiang J, A Porphyrin-pyranine Dyad for Ratiometric Fluorescent Sensing of
Intracellular pH, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2020),
doi: https://doi.org/10.1016/j.jphotochem.2020.112524

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A Porphyrin-pyranine Dyad for Ratiometric
Fluorescent Sensing of Intracellular pH
JinghuiZhang,† Mengliang Zhu,† Jiawen Cui,‡ Chiming Wang,† Ziwen Zhou,† Tianhang
Wang,†Lei Gong,† Chaorui Su,† Dongdong Qi,†Yongzhong Bian,*,†Hongwu Du,*,‡ and
Jianzhuang Jiang*,†
†Beijing Key Laboratory for Science and Application of Functional Molecular and Crystalline
Materials, Department of Chemistry, University of Science and Technology Beijing, Beijing
100083, China
‡Department of Biology, University of Science and Technology Beijing, Beijing 100083, China
*To whom correspondence should be addressed. E-mail: yzbian@ustb.edu.cn (Y.B.),
hongwudu@ustb.edu.cn (H.D.), jianzhuang@ustb.edu.cn(J.J.)
Graphical abstract
The FRET-based porphyrin-pyranine dyad 1 exhibited sensitive and reversible emission ratio
(F660/F435) response in a dynamic pH range of 2.6-8.6, and thus was applied for pHi calibration
by fluorescence ratio imaging in living cells.
1

Highlights
 A FRET-based porphyrin-pyranine dyad for ratiometric fluorescence sensing was
developed.
 The dyad shows sensitive fluorescent ratio (F660/F435) responses to pH in PBS buffer.
 The dyad can be applied for pHi calibration by fluorescence ratio imaging in living cells.
ABSTRACT
The detection of intracellular pH (pHi) by fluorescence imaging is highly desired for cell biology
and medical science. In this study, we developed a FRET-based porphyrin-pyranine dyad (Por-
Py 1) composed of a pyranine moiety as the energy donor and a porphyrin moiety as the pH-
responsive energy acceptor for ratiometric fluorescent sensing of pHi. Por-Py 1 displays a 95-
2

fold fluorescent intensity ratio (F660/F435) enhancement upon pH change from 2.59 to 8.58 in PBS
buffer. The two pKa values (pKa1 = 3.92 and pKa2 = 6.46) obtained can be assigned to the
successive and reversible protonation/deprotonation of the meso-pyridyl groups and inner
pyrrole units of the porphyrin moiety, respectively. Por-Py 1 also exhibits good cell permeability,
lysosome-targeting ability, and low cytotoxicity, ensuring its further application for pHi
calibration by fluorescence ratio imaging in living cells.
Keywords: Porphyrin, Pyranine, pH sensor, Ratiometric fluorescent
sensor,Fluorescenceresonance energy transfer
1. Introduction
Intracellular pH (pHi) is a key factor for modulating fundamental physiological functions of
cells[1]. Abnormal pHi is strongly coupled with cellular dysfunction and usually indicative of
various disease states such as cancer, diabetes, and inflammation[2].The accurate measurement
of pHi is thus highly desired for the study of related physiological and pathological
processes[3,4].
Fluorescent imaging is a promising technique for the detection of pHi due to the advantages in
sensitivity, selectivity, spatiotemporal resolution, and biocompatibility[5,6]. In recent years,
great efforts have been invested in the development of fluorescent molecules capable of
quantifying pH values in living cells[7-9]. However, most of the reported fluorescent sensors are
based on pH-dependent switching on or off of a single emission band, which are susceptible to
interference from the fluctuation of conditions and background signals. Alternatively, ratiometric
fluorescent pH sensors employ the ratio of two distinct emission bands[10], which can provide
3

self-calibration by dual-channel detection, thus minimize possible interference and perform
precise pH analysis against environmental and instrumental factors[11-14]. Therefore, the design
of ratiometric fluorescent sensors for pHi detection is of great importance while it remains
challenging, because the ratiometric fluorescent sensing performance need to be further
improved for practical applications.
Fluorescence resonance energy transfer (FRET) is a useful strategy for ratiometric fluorescent
sensing and bioimaging[15]. For a typical FRET donor-acceptor system, the intramolecularFRET
efficiency can be modulated in an analyte-dependent manner, leading to sensitive emission ratio
changes in response to a target analyte[16,17]. Herein, we report the rational design of
aporphyrin-pyranine dyad (Por-Py 1) for ratiometric fluorescent sensing of pHi (Scheme1).
Porphyrins (Pors) are suitable candidates forfluorescent sensing and imaging due to the superior
photophysical and biocompatible properties[18-23]. In particular, meso-pyridyl substituted
porphyrins can undergo reversible and stepwise protonation of the meso-pyridyl groups and the
inner pyrrole units in a wide pH range[24,25], which may offer the bases for pH-dependent
absorption and emission properties. However, due to the poor water solubility, meso-pyridyl
substituted porphyrins cannot be used directly for pH sensing in aqueous media or biological
samples. On the other hand, pyranine (Py) is known as a highly water-soluble fluorescent
dye[26], while the lack of cell permeability hinders its practical use in pHi detection[7]. In this
study, the FRET dyad Por-Py1 was formulated by a pyranine unit as the energy donor and a
meso-4-pyridyl substituted porphyrin unit as the acceptor. Notably, the fluorescent emission of
Py overlaps well with the Soret absorption of Por in 400-450 nm range, which allows for an
efficient FRET process. Furthermore, the hydrophilic Py can improve aqueous solubility, and the
hydrophobic Por can increase cell permeability, both are significant for the practical application
4

in pHi detection. To our knowledge, Por-Py 1 represents the first example of covalently linked
porphyrin-pyranine system, which was found exhibiting sensitive and reversible emission ratio
response within a dynamic pH range of 2.59-8.58 in PBS buffer, and was also applied for pHi
calibration by fluorescence ratio imaging in living cells.
Scheme 1.Schematic molecular structures of Por-Py 1 and the reference compounds 2 and 3.
2. Results and discussion
2.1 Synthesis of Por-Py 1
The porphyrin-pyranine dyad Por-Py1 was synthesized via the Williamson's coupling reaction
between Py 2 and the 4-hydroxyphenyl substituted tri(4-pyridyl) porphyrin (Por 4). All of the
new compounds were adequately characterized (Scheme S1 and Figures S1-S5).
5

Figure 1. The electronic absorption (black line) and emission spectra (red dotted line) of Por-Py
1 in DMF (2.0 ×10-6 M) with excitation at 370 nm.
2.2 Spectroscopic properties
The electronic absorption spectrum of Por-Py 1 shows the characteristic Soret band (ca. 420
nm) and Q bands (500-650 nm) of the Por moiety, in addition to the relatively weak absorptions
of the Py moiety (280-400 nm), which resembles the linear superimposition of those of
compounds Py 2 and Por 3, indicating the absence of strong electronic interaction between the
Por and Py moieties in the ground state (Figures 1 and S6, Table S1). Upon excitation at 370 nm,
where the Por and Py moieties have comparable absorbance, Por-Py 1 gives a strong band in
blue region (400-500 nm) and a moderate one in near-infrared (NIR) region (620-720 nm). The
two well resolved emission bands are feasible for dual-channel detection. The fluorescence
spectrum of Por-Py 1 is similar to that of the reference Por 3 (Figures 1 andS6, Table S1),
therefore the blue and NIR bands can be assigned to S2→S0 and S1→S0 emissions of the Por
moiety respectively[27,28]. As expected, the emission of the Py moiety in Por-Py 1 is
significantly quenched due to the efficient intramolecular FRET from the energy donor (Py) to
the energy acceptor (Por) (Figure S7). The energy transfer efficiency (ηEET) was estimated to be
6

99%[29], which is reasonable given the effective spectral overlap between the emission of Py
and the absorption of Por (Figure S10) as well as the short distance between the two fluorophores
in Por-Py 1 (Figure S11)[30]. Notably, as the emission of Py is partially overlapped with the
S2→S0 emission of Por, the residual emission of the Py moiety may also contribute to the blue
band (400-500 nm) of the dyad, leading to slightly different emission patterns for Por-Py 1 and
Por 3 (Figures 1 and S6).
The fluorescence lifetime of Por-Py 1 was also measured in DMF with excitation at 375 nm,
and compared with those of Py 2 and Por 3 (Figures S8 and S9, Table S1). The decay of
fluorescence at 405 nm (S2→S0 emission of Por 3) exhibits a biexponential feature of 1.6 ns
(population ratio 59.6%) and 9.7 ns (40.4%), which is slightly disturbed by the formation of Por-
Py 1, with τ1 = 1.7 ns (49.2%) and τ2 = 7.2 ns (50.8%). The decay of fluorescence at 650 nm
(S1→S0 emission of Por 3) is monoexponential with a lifetime of 10.0 ns, which is unaltered by
the formation of Por-Py 1. For Py 2, the decay of fluorescence at 405 nm is also
monoexponential with a lifetime of 3.4 ns. However, because this emission is highly quenched
and the residual emission is overlapped with the strong S2→S0 emission of the acceptor (Por),
the decay profiles of the donor (Py) in Por-Py 1 have not been obtained.
2.3. Ratiometric fluorescent pH sensing behavior in solution.
The optical response of Por-Py 1 to pH was evaluated in PBS buffer solution (Figure 2). With
decreasing the pH value from 8.58 to 2.59, the Soret band of Por moiety decreases in intensity
and undergoes bathochromic shift from 419 to 450 nm gradually. Two successive isosbestic
points are observed at 432 and 445 nm, corresponding to the stepwise protonation of the pyridyl
and pyrrolyl nitrogen atoms of Por moiety in this pH range[24,25]. Changes in the Q bands of
7

Por moiety are also weakly visible, such as diminishing at 520 nm and raising at 660 nm (Figure
2a).
Figure 2. Optical responses of Por-Py 1 (1.0 ×10-6 M) in PBS buffer at various pH values in the
range of 2.6-8.6. (a) Absorption spectra; (b) Emission spectra (λex = 370 nm); (c) Fluorescence
intensity ratio (F660/F435) as a function of pH value; (d) Fluorescence intensity ratio (F660/F435)
changes as a rectilinear function of pH (5.4-7.5).
The fluorescent changes of Por-Py 1 are also distinct along with pH fluctuation (Figure 2b).
The near-infrared emission band at 660 nm quenches dramatically as the pH value decreases
from 8.58 to 2.59, while the blue emission band at 435 nm only slightly decreases concomitantly,
8

leading to a 95-fold intensity ratio (F660/F435) change from 1.73 (pH = 8.58) to 0.018 (pH = 2.59).
Significantly, plotting the ratio (F660/F435) versus pH gives a clear two-stage fitting curve (Figure
2c), which is consistent with the stepwise protonation of meso-pyridyl substituted
porphyrins[24,25]. By taking the first derivative of the fitting curve, two apparent pKa values can
be acquired for Por-Py 1, in which pKa2 (6.46) is assigned to the protonation/deprotonation of
meso-pyridyl groups, and pKa1 (3.92) to that of inner pyrrole units[31]. Two linear responsive pH
ranges of 5.4-7.5 and 3.3-4.5 are observed around the two pKa values (Figures 2dandS12), where
the former one is suitable for quantitative detection inphysiological pH range.
Scheme 2. Proposed pH sensing mechanism of Por-Py 1.
Furthermore, the fluorescence emission and the intensity ratio (F660/F435) of Por-Py 1 show
reversible variations as pH was switched repeatedly between 4.0-8.0 (Figure S13), which is
believed to be due to the reversible protonation/deprotonation processes of the Por moiety, as the
Py moiety is almost unaffected in this pH range (Figure S14)[32]. Accordingly, the mechanism
of the pH-dependent fluorescent response can be proposed to the reversible and stepwise
protonation/deprotonation of the Por moiety(Scheme 2), which induces the reversible
quenching/recovery of the red emission band (660 nm) while the blue emission band (435 nm) is
barely affected, affording the sensitive emission ratio (F660/F435) response to pH change.
9

2.3 Cellular pH sensing behavior
Figure 3.Confocal fluorescence images for intracellular localization of Por-Py 1. A549 cells
were treated with 1 (7.5 μM) for 30 min and further stained with DioC18(3) (4 μM), Mito
Tracker™ Green FM (100 nM), or LysoTracker™ Green DND-26 (100 nM) for another 20 min.
Green Channel: λex = 488 nm, λem = 500-600 nm; Red Channel: λex = 405 nm, λem = 620-750 nm.
The last column shows the corresponding intensity profiles within the linear region of interest
(ROI, given by blue lines in the merged images) across cells.
Based on the above ratiometric fluorescent properties, Por-Py 1 was applied to living cells and
monitored by confocal fluorescence microscopy. After incubation with Por-Py 1 for 30 min,
A549 cells exhibited strong fluorescence in the red channel (620-700 nm), suggesting the
10

favorable cell permeability (Figure S15). The co-localization experiments were performed by
costaining with commercial cytomembrane probe Dio, Mito-Tracker, and Lyso Tracker (Figure
3). The red channel image of Por-Py 1 overlaps well with the green channel image of Lyso
Tracker, and the variations of fluorescence intensity in the linear region of interest (ROI) are
nearly synchronous for the two channels. In comparison, the red fluorescence of Por-Py 1 cannot
overlap the green fluorescence of Dio or Mito-Tracker, thus demonstrating the specific
localization of Por-Py 1 to lysosomes[33].MTT assay was performed to evaluate the cytotoxicity.
The cellular viability was not significantly changed upon treatment with Por-Py 1 (up to 20.0
μM)for 24 h, indicating the low cytotoxicity to A549 cells (Figure S16).
Figure 4. Fluorescence images of A549 cells. The cells were incubated with Por-Py 1 (7.5 μM in
DMEM) for 30 min, and then treated with the calibration buffers at various pH values (4.0, 6.0
and 8.0) for 30 minat 37℃. (a) Red channel (620-700 nm); (b) Blue channel (400-500 nm); (c)
Ratio images. Scale bar = 20 μm.
11

For quantifying the intracellular pH, A549 cells were stained with Por-Py 1 and treated with
buffers (containing 10 μM nigericin and 5 μM monensin) to homogenize the pH values to 4.0,
6.0 and 8.0, respectively. As increasing the pH from 4.0 to 8.0, the red intracellular fluorescence
enhanced obviously (Figure 4a), while the blue one remained almost unchanged (Figure 4b),
resulting in the significant enhancement of ratio images (Figure 4c). The intracellular pH
calibration profile was thus obtained by plotting the average intensity ratio of the red to blue
channels (Rred/blue) versus pH (Figure S17). It is worth noting that the fluorescent ratio response
in A549 cells (Rred/blue) is lower than that in PBS buffer (F660/F435 in Figure 2d), which may due
to the following reasons: 1) the microenvironment of cells is complicated and not uniform, 2) the
dye molecules may aggregate in cells, and 3) the pseudo-ratio values (Rred/blue) were generated
from the ratio images of cells by an image processing software (ImageJ), which are different
from the spectral properties in solution. However, the variation trend of Rred/blue in the cells is
consistent with the sensitive fluorescent ratio responses in solution, suggesting the potential of
Por-Py 1 for ratiometric fluorescent sensing of pH in living cells.
3. Conclusion
In summary, a porphyrin-pyranine dyad has been developed for the ratiometric fluorescent
detection of pHi. The dyad can produce two well resolved emission bands in blue and NIR
regions for dual-channel detection. The intensity ratio (F660/F435) of the two emission bands is
highly sensitive to pH in PBS buffer solution, leading to a 95-fold change in a wide pH range. By
plotting the ratio (F660/F435) versus pH values, two successive apparent pKa values are obtained,
corresponding to the stepwise and reversible protonation/deprotonation of meso-pyridyl groups
12

and inner pyrrolyl units, respectively, of the porphyrin moiety. The dyad also displays superior
biocompatible properties, and thus can be a promising candidate for pHi sensing by fluorescence
ratio imaging.
4. Experimental section
4.1 General Experimental Details
Column chromatography was carried out on silica gel (Qingdao Ocean Chemicals, 200-300
mesh) with the indicated eluents. DMSO was freshly distilled with CaH2 under nitrogen
atmosphere. All other reagents and solvents were used as received without further purification
unless noted otherwise.
1H NMR spectra were recorded on a Bruker DPX 400 MHz spectrometer in DMSO-d6 and the
chemical shifts were reported relative to internal SiMe4. MALDI-FT mass spectra were
measured on a Solar IX Fourier transform (FT) ion cyclotron resonance mass spectrometer, and
MALDI-TOF mass spectra were measured on a Bruker MicroflexTM LRF spectrometer with
dithranol as the matrix. Elemental analyses were performed on an Elementar Vario MICRO
CUBE elemental analyzer. Electronic absorption spectra were recorded on a Lambda 750
spectrophotometer. Steady-state fluorescence spectroscopic studies were performed on a Hitachi
F4500 fluorophotometer. The time-resolved fluorescence spectra were acquired on a FLS980
spectrofluorimeter (Edinburgh) with excitation at 375 nm by a EPL laser. The pH measurements
were carried out with a Mettler-Toledo FE20 pH meter. Fluorescence imaging experiments were
performed at 37°C on a Nikon A1RSi+ confocal laser scanning microscope through 100× oil
immersion objective lens or a Leica SP8 confocal microscope through 40× objective lens. The
cell cytotoxicity was measured using MTT assay on a Tecan SunriseTM microplate reader.
13

4.2 Synthesis
4.2.1 Synthesis of 5-(4-methoxyphenyl)-10,15,20-tri-(4-pyridyl)-21,23H-porphyrin (Por 3)
Freshly distilled pyrrole (14.06 mL, 0.15 mol) and 4-pyridinecarboxaldehyde (22.5 mL, 0.125
mol) were dissolved in propionic acid (700 mL). After heated to refluxing, 4-
methoxybenzaldehyde (3.0 mL, 0.025 mol) was added dropwise in 10 min. After further
refluxing for 40 min, the mixture was cooled to room temperature and then propionic acid was
removed under reduced pressure. The residual was extracted by CH2Cl2 and then washed with
saturated aqueous NaHCO3 and water. The organic phase was dried over anhydrous magnesium
sulfate and concentrated at reduced pressure. The crude product was purified by silica gel
column with CH2Cl2/CH3OH (100/3, in v/v) as the eluent, where the third fraction was collected
and evaporated to give Por 1 as purple powder (800 mg, 5%). 1H NMR (400 MHz, DMSO-d6,
δ): 9.05 (d, J = 4.4Hz, 6 H), 8.93-8.89 (m, 8 H), 8.27 (s, 6 H), 8.15 (d, J = 8.0 Hz, 2 H), 7.41 (d,
J = 8.4 Hz, 2 H), 4.06 (s, 3 H), -3.01 (s, 2 H). MALDI-TOF-MS: m/z calcd for C42H29N7O
[M+H]+ 648.25; found 648.27.
4.2.2. Synthesis of 5-(4-hydroxyphenyl)-10,15,20-tri-(4-pyridyl)-21,23H-porphyrin (Por 4)
The above obtained Por 3 (788 mg, 1.21 mmol) was mixed with excess pyridine hydrochloride
(5 g) and heated to 170℃. After stirring at this temperature in the nitrogen atmosphere for 12 h,
the mixture was cooled to room temperature and washed with saturated aqueous NaHCO3 and
water. The residual was extracted by CHCl3 and dried over anhydrous Na2SO4. Then the organic
phrase was evaporated to dryness under reduced pressure. The crude product was purified by
silica gel column with CH2Cl2/CH3OH (25/1, v/v) as the eluent to yield pure Por 4 as red powder
(250 mg, 33%).1H NMR (400 MHz, DMSO-d6, δ): 9.29 (d, J = 5.2 Hz, 6 H), 9.03-8.99 (m, 8 H),
14

8.71-8.67 (m, 6 H), 8.02 (d, J = 8.0 Hz, 2 H), 7.25 (d, J = 8.4 Hz, 2 H), -3.01 (s, 2 H). MALDI-
TOF-MS: m/z calcd for C41H27N7O [M+H]+ 634.24; found 634.62.
4.2.3 Synthesis of 8-(4-bromobutoxy) pyrene-1,3,6-trisulfonatesodium (Py 2)
To a refluxing solution of 8-hydroxypyrene-1,3,6-trisulfonate (500 mg, 0.954 mmol) in
CH3OH (50 mL) was added 1,4-dibromobutane (2.28 mL, 19.08 mmol) and N,N-
diisopropylethylamine (1.65 mL, 10 mmol), and the resulting mixture was refluxed with stirring
for 5 days. During the reaction process, N,N-diisopropylethylamine was supplemented once the
mixture turned fluorescent green. After cooled to room temperature, the solution was filtered and
concentrated under reduced pressure. Further purification by silica gel column with
CHCl3/CH3OH/H2O (7/4/1, v/v/v) afforded Py 2 as yellow solid (175 mg, 28%).1H NMR (400
MHz, DMSO-d6, δ): 9.12 (d, J = 9.6 Hz, 2 H), 9.04-9.02 (m, 2 H), 9.02 (s, 1 H), 8.94 (d, J = 10.0
Hz, 1 H), 8.37 (d, J = 9.6 Hz, 1 H), 8.19 (s, 1 H), 4.41 (t, J = 6.0 Hz, 2 H), 3.37 (t, J = 6.0 Hz, 2
H), 2.19-2.10 (m, 4 H).
4.2.4. Synthesis of Por-Py 1
In the presence of anhydrous cesium carbonate (300 mg), a solution of Por 4 (60 mg, 0.09
mol) and Py 2 (67 mg, 0.11mol) in dry DMSO (5ml) was heated to 120℃. After 12 h, the
reaction mixture was cooled to room temperature and filtered, and DMSO was removed under
reduced pressure. The crude product was purified by silica gel column with CH2Cl2/CH3OH/H2O
(7/3/0.5, v/v/v) as the eluent to yield the target compound Por-Py 1 (30 mg, 26%). 1H NMR (400
MHz, DMSO-d6, δ): 9.19 (d, J = 9.6 Hz, 1 H), 9.08-9.04 (m, 8 H), 8.98-8.97 (m, 3 H), 8.89 (s, 6
H,), 8.50 (d, J = 9.6 Hz, 1 H), 8.31 (s, 1 H), 8.28 (d, J = 4.0 Hz, 6 H), 8.13 (d, J = 8.4 Hz, 2 H),
7.46 (d, J = 8.0 Hz, 2 H), 4.57 (t, J = 4.8 Hz, 2 H), 4.46 (t, J = 4.8 Hz, 2 H), 2.30 (m, 4 H), -3.01
15

(s, 2H). MALDI-FT-MS: m/z calcd for C61H43N7O11S3 [M+H]+ 1146.23; found 1146.23. Anal.
Calc. For C61H43N7O11S3: C, 51.7%; H, 3.52%; N, 6.68%; S, 6.55%. Found: C, 51.8%; H,
3.85%; N, 6.76%; S, 6.74%.
4.3. Ratiometric Fluorescence Determination
250 μL Por-Py 1 solution (1.0×10-3 M in DMSO) were added to 250 mL PBS buffer to obtain
the working solution (1.0×10-6 M). The bulk solution was titrated with NaOH (6.0 M) and HCl
(6.0 or 12.0 M) to various pH values. The pH values were measured by Mettler-Toledo FE20
pH-meter. At each pH, the solution was transferred to a quartz cell of 1 cm optical length to
measure absorption and fluorescence (λex = 370 nm) spectra. The ratio signal was calculated
from the fluorescence intensities at 660 nm and 435 nm.
4.4. Confocal Fluorescence Imaging
The A549 cells were grown on glass-bottom culture dishes in Dulbecco's modified eagle
media supplemented with 10% (v/v) fetal bovine serum, penicillin (100 μg/mL), and
streptomycin (100 μg/mL) at 37°C in a 5% CO2 incubator. Cells were treated with Por-Py 1 (7.5
μM in DMEM) for 30 min and then washed with PBS solution to remove the excess Por-Py 1.
Fluorescence imaging experiments were performed at 37°C on a Nikon A1RSi+ confocal laser
scanning microscope with live cell imaging system and excitation at 405 nm through 100× oil
immersion objective lens. The fluorescence was collected in the range of 620-700 nm (red
channel). Image processing and analysis were performed on Image-J.
4.5. Cell localization Experiment
16

A549 cells were incubated with Por-Py 1 (7.5 μM in DMEM, with 0.75% DMSO) at 37°C for
30 min, and then stained with DioC18(3) (4 μM), Mito Tracker™ Green FM (100 nM), or Lyso
Tracker™ Green DND-26 (100 nM) for another 20 min. Before fluorescence imaging, the
adherent cells were further washed twice with PBS (pH 7.4) to remove the excess dyes.
Fluorescence imaging experiments were performed on a Leica SP8 confocal microscope through
40× objective lens. Two color channels were used for these experiments: red channel (λex = 405
nm, λem = 620-750 nm) for Por-Py 1, and green channel (λex = 488 nm, λem = 500-600 nm) for
DioC18(3), Mito Tracker and Lyso Tracker.
4.6. Intracellular pH Calibration
For the ratiometric pH calibration in live A549 cells, the cells were stained with DMEM
containing Por-Py 1 (7.5 μM) for 30 min at 37℃ in a 5% CO2 incubator, then the adherent cells
were washed with PBS (pH 7.4) to remove the excess Por-Py1. The cells were further incubated
(15 min) with high K+ buffers of different pH values (4.0, 6.0, 8.0) in the presence of 10 μM
nigericin and 5 μM monensin. Fluorescence imaging experiments were performed at 37°C on a
Nikon A1RSi+ confocal laser scanning microscope through 100× oil immersion objective lens.
Upon the excitation of 405 nm, the blue channel images (400-500 nm) and the red channel
images (620-700 nm) were collected, respectively. Pseudo-colour ratiometric images were
obtained by mediating the blue and red channel images at the same pH value. The pH calibration
was finally obtained based on the average ratio values.All data were expressed as mean 
standard deviation. Image processing and analysis were performed on Image-J.
Declaration of interests
17

The author declare no conflict of interests
Author contributions
Jinghui Zhang: Investigation; Conceptualization; Writing-Original Draft.
Mengliang Zhu: Conceptualization; Formal analysis.
Ziwen Zhou and Tianhang Wang: Investigation; Data curation
Chiming Wang, Lei Gong, and Dongdong Qi: Investigation; Data curation.
Yongzhong Bian: Conceptualization; Supervision; Validation; Writing-review & editing
Hongwu Du: Investigation; Resource for A549 cells.
Jianzhuang Jiang: Funding acquisition; Project administration; Validation; Writing-review &
editing.
Acknowledgments
Financial support from the National Natural Science Foundation of China (21631003, 21671017
and 31371203), Beijing Natural Science Foundation (2202028) and Fundamental Research
Funds for the Central Universities (No. FRF-BD-19-013A) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://.
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