Visualizing semipermeability of the cell membrane using a pH-responsive ratiometric AIEgen

Article abstract of DOI:10.1039/d0sc02097d

In clinical chemotherapy, some basic drugs cannot enter the hydrophobic cell membrane because of ionization in the acidic tumor microenvironment, a phenomenon known as ion trapping. In this study, we developed a method to visualize this ion trapping phenomenon by utilizing a pH-responsive ratiometric AIEgen, dihydro berberine (dhBBR). By observing the intracellular fluorescence ofdhBBR, we found that non-ionizeddhBBRcan enter cells more easily than ionized forms, which is in accordance with the concept of ion trapping. In addition,dhBBRshows superior anti-photobleaching ability toCurcuminthanks to its AIE properties. These results suggest thatdhBBRcan serve as a bioprobe for ion trapping.

Full text of DOI:10.1039/d0sc02097d

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DOI: 10.1039/D0SC02097D
ARTICLE
Visualizing Semipermeability of Cell Membrane by a pH-
responsive Ratiometric AIEgen
Yuan Gu,ab Zheng Zhao,ab Guangle Niu,ab Han Zhang,c Yiming Wang,ab Ryan T. K. Kwok,ab Jacky
Received 00th January 20xx,
Accepted 00th January 20xx
W. Y. Lam,ab and Ben Zhong Tang*abc
DOI: 10.1039/x0xx00000x
In clinical chemotherapy, some basic drugs cannot enter the hydrophobic cell membrane because of ionization in acidic
tumor microenvironment, a phenomenon known as ion trapping. In this study, we developed a method to visualize this ion
trapping phenomenon by utilizing a pH-responsive ratiometric AIEgen, dihydro berberine (dhBBR). By observing the
intracellular fluorescence of dhBBR, we found that non-ionized dhBBR can enter cells easier than ionized forms, which is in
accordance with the concept of ion trapping. In addition, dhBBR shows superior anti-photobleaching ability than Curcumin
thanks to its AIE property. These results suggest that dhBBR can serve as a bioprobe for ion trapping.
Fluorescent technology, with their charming merits of simplicity,
high sensitivity, and low background noise, is becoming more
and more popular in the biomedical research.15 Thanks to the
Introduction
All living cells must exchange materials with their extracellular
environment in order to keep alive.1,2 The process of substances
entering and out of cells are controlled by cell membrane, a
biological membrane consisting of a lipid bilayer with proteins
embedding in, which separates interior of cells from their external
environment.3,4 Due to the semipermeable nature of lipid bilayer,
cell membrane is permeable to non-ionized (fat-soluble) molecules,
while the permeability to ionized (water-soluble) molecules is very
limited, a phenomenon commonly known as ion trapping.5 Partial
failure in the cancer chemotherapy of some basic drugs, such as vinca
alkaloids and anthracyclines, can be ascribed to ion trapping. The
acidic tumor microenvironment prevents the ionized alkaline drugs
from accumulating in cancer cells.5-9 Given that, the study of cell
membrane’s semipermeability, especially ion trapping, is not only
fundamentally interesting, but also valuable in improving the clinical
chemotherapeutic efficacy.
Ion trapping is most widely studied by HPLC, a well-developed
technology.5,9-12 Although HPLC enjoy the advantages of high
sensitivity and selectivity, it usually brings about thorny issues like
high equipment cost, complexity, complicated sample processing,
and long runtime. Worse more, the biological sample usually needs
to be isolated and homogenized, which makes in situ monitoring
biological process impossible.13,14
enthusiastic endeavors made by scientists, a lot of fluorescent
bioprobes have been developed for various applications.16-19
Drugs with inherent fluorescence make real-time in situ tracking
of drug molecules in vivo or in vitro possible, which is of critical
importance in pharmaceutical research. However, so far,
fluorescent drug as probe for monitoring ion trapping has
scarcely been reported in spite of its significance in studying the
drug delivery and drug distribution in body. This is partly
because it is still difficult to find a drug which shows pH-
responsive fluorescence. And it is even more challenging to seek
a drug which has different emission behaviors between its non-
ionized and ionized forms. What’s more, fluorescent probes
usually suffer from aggregation-caused quenching (ACQ)
effect.20 In 2001, Tang and co-workers discovered aggregation-
induced emission (AIE), which is directly opposite to ACQ.21
Since then, a variety of AIEgens have been developed for many
advanced applications.22,23 Recently, Tang have put forward
natural resources as a new source to explore AIEgens.24 AIEgens,
usually obtained from herbal plants, animals, and other natural
resources, have a lot of unique advantages, such as synthesis-
free, environmental friendly, pharmaceutically active, etc.24-26
Previously, we have reported that Berberine Chloride, a natural
isoquinoline isolated from Chinese herbal plants, is a rotor-free
AIEgen.24 In this study, dihydro Berberine (dhBBR) that
converted from Berberine Chloride by gut microorganisms was
found to be an AIEgen with pH sensitivity. More importantly,
the highly similar molecular structure of dhBBR as BBR enables
it work as a suitable drug-like probe for visualizing cell
membrane’s semipermeability thanks to its ratiometric
fluorescence.
a. Department of Chemical and Biological Engineering, Department of Chemistry, the
Hong Kong Branch of Chinese National Engineering Research Center for Tissue
Restoration and Reconstruction and Institute for Advanced Study, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
999077, China.
b. HKUST- Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area Hi-tech
Park, Nanshan, Shenzhen 518057 China.
c. Center for Aggregation-Induced Emission, SCUT-HKUST Joint Research Institute,
State Key Laboratory of Luminescent Materials and Devices, South China University
of Technology, Guangzhou 510640, China.
† Electronic Supplementary Information (ESI) available: Materials and Methods; 1H
NMR and HRMS spectra of compounds; Photophysical data and Imaging data.
Results and discussion
Synthesis and photophysical properties
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vibration in determining the photophysical properties of dhBBR, viscosity-
View Article Online
and temperature-dependent fluorescence cDhOanI:g1e0s.10o3f9/dDh0BSBCR020w9e7rDe
investigated.AsshowninFig.S4(AandB),dhBBRshowsstrongeremission
in viscous solvents or at low temperature because the intramolecular
vibration is suppressed.
pH-dependent ratiometric fluorescent response
The nitrogen atom in the molecular backbone of dhBBR is speculated
to be protonated in acid.31,32 As shown in Fig. 3A, dhBBR exhibits
obvious pH-dependent fluorescence. Adding acid into dhBBR causes
a gradual blue shift of dhBBR’s emission from 511 nm to 454 nm as
shown in Fig. 3A. To understand dhBBR’s pH-dependent fluorescence,
absorption titration experiment is performed. As shown in Fig. S5,
there is a red shift of the maximum absorption band of dhBBR from
350 nm to 426 nm when the pH of the buffer was changed from 2 to
Fig. 1 (A) Synthetic route to dhBBR. (B) Quantum Yield of dhBBR in DMSO solution (10
μM), single crystal, and powder states. Excitation wavelength: 365 nm. (C) Time-resolved
emission decay curves of dhBBR in DMSO solution, single crystal, and powder states.
Solution concentration: 10 µM; Excitation wavelength: 365 nm.
1
9. H NMR titration study was performed by adding trifluoroacetic
acid (TFA) into dhBBR. As shown in Fig. S2, there are downfield shifts
of the isoquinoline protons induced by TFA. The new peak at 8.0
suggested that the nitrogen atom in the isoquinoline is protonated.
pH titration experiment was then conducted. The fluorescence
maximum of dhBBR exhibited a gradual red shift when the pH
increased from 1 to 12 (Fig. 3B). More importantly, dhBBR showed
an excellent linearity of I511/I454 in the pH range of 7-10 with a pKa
value of 8.4 (Fig. 3C), which indicated that dhBBR can serve as a
dhBBR was synthesized according to the route shown in Fig. 1A with
86% yield.27 The purity of the product was confirmed by 1H NMR as
well as high resolution mass spectroscopy (HRMS) (Fig. S1, S3 in ESI).
The photoluminescence quantum yield (PLQY) of dhBBR in DMSO
solution and crystal states were measured to be 7.9% and 28.8%,
respectively (Fig. 1B). The results indicate that dhBBR is a strong
solid-state emitter. The fluorescence lifetime of dhBBR in the crystal
state (4.68 ns) is longer than that in the solution state (1.1 ns) (Fig.
1C). In addition, from solution to crystal state, the non-radiative
decay rate decreases about 5.5 times (Knr,soln=8.37 × 108 s-1,
Knr,crystal=1.52 × 108 s-1), which should be responsible for the AIE
property of dhBBR.
ratiometric pH probe. Furthermore, dhBBR showed
a good
reversibility between pH 2 and pH 12 as shown in Fig. 3D. In addition,
the intensity ratios (I511/I454) of dhBBR in pH 3 and pH 10 buffers keep
nearly unchanged after 10 min (Fig. S6). The fluorescence response
of dhBBR towards different interfering species was evaluated (Fig.
3E). The intensity ratio (I511/I454) was neglectively affected by
common metal ions (Na+, K+, Ca2+, Mg2+, Fe2+, Cu2+, Zn2+, Mn2+, Al3+,
Pb2+, 0.1 mM for Na+, K+, Ca2+, Mg2+, 0.01 mM for Fe2+, Cu2+, Zn2+,
Mechanism study
To have a clear picture on the photophysical properties of dhBBR.
The single-crystal structure of dhBBR was analyzed.28 As shown in Fig.
2, the conformation of dhBBR in crystal is non-planar with a 27.94o
twisted angle, which indicates that intramolecular vibration is
possible. In addition, the intermolecular distance of adjacent dhBBR
molecule aligned in parallel was measured to be 3.771 Å, which
exceeds the effective π–π stacking distance (3.5 Å) to quench the
fluorescence.29 What’s more, multiple intermolecular interactions (2.408-
2.888 Å in distance) contribute to rigidify the molecular conformation
which makes dhBBR highly emissive in the crystal. Thus, the brighter
emission of dhBBR in crystal than that in solution can possibly be
explained by the restriction of intramolecular vibration which suppresses
the non-radiative decay pathway.30 To further clarify the intramolecular
Fig.
2 Single crystal packing of dhBBR. (A) Intramolecular torsional angle. (B)
Intermolecular distance of adjacent molecules. (C) Intermolecular interactions.
2 | J. Name., 2012, 00, 1-3
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Fig. 3 (A) Schematic illustration of dhBBR’s fluorescent response to pH Change. (B)
Emission spectra of dhBBR in the PBS buffer solutions with different pH values. [dhBBR]
= 10 μM. (C) Plot of I511/I454 versus pH. I511 and I454 denote the emission intensities of the
solution at 511 and 454 nm, respectively. Excitation wavelength: 365 nm. (D)
Fluorescence reversibility of dhBBR in PBS buffer between pH 2.0 and pH 12.0. [dhBBR]
= 10 μM. (E) Ratiometric fluorescent responses of dhBBR (10 μM) to different potential
interfering agents in pH 3.0 (red column) and 10.0 (black column) PBS buffer solutions:
pH to be constant. Instead, we incubated cells VwiewitAhrticdlehOBnBliRne-
containing PBS buffer of varied pH (pH from 4 to 7.4) for a period.
DOI: 10.1039/D0SC02097D
Cells were then imaged under confocal microscopy. It was intriguing
to notice that A549 cells incubated with dhBBR-containing PBS of a
pH range of 4-6 show almost no emission from both blue and green
channels (blue channel: 400-460 nm; green channel: 500-600 nm)
(Fig. 4A). In contrast, obvious green fluorescence was observed
inside cells when A549 cells were incubated with dhBBR-containing
PBS buffer of a pH 7.4 (Fig. 4A). Also, increasing the pH of the PBS
buffer from 4 to 7.4 can also induced a gradual increase of emission
ratio (IGreen / IBlue) (Fig. 4B). Similar results were also observed in HEK
293T cells (Fig. 4C and D). Additionally, different concentrations of
dhBBR were used and similar results were obtained in HeLa cells (Fig.
S8). This “acid out, base in” phenomenon can be ascribed to the
semipermeability of cell membrane as mentioned above. Due to the
hydrophobic nature of cell membrane, it makes non-ionized dhBBR
easier to enter into cells compared to ionized forms, a phenomenon
known as “ion trapping”, which well explains why incubating cells
with dhBBR-containing PBS of higher pH value causes stronger
intracellular fluorescence than dhBBR-containing PBS with lower pH
value. Photostability is an important parameter in evaluating a bio-
probe’s anti-photobleaching ability. As shown in Fig. 5, more than
70% of dhBBR’s fluorescence is retained even after 200 seconds of
irradiation, while less than 40% of curcumin’s fluorescence is
retained after the same irradiation time, suggesting that dhBBR has
superior anti-photobleaching ability than curcumin thanks to its AIE
property.
(0) control; (1) Na+; (2) K+; (3) Ca2+; (4) Mg2+; (5) Fe2+; (6) Cu2+; (7) Zn2+; (8) Mn2+; (9) Al3+
;
(10) Pb2+; (11) Ac-; (12) SO42-; (13) NO3 ; (14) PO42-; (15) S2O32-; (16) CO32-; (17) Phe; (18)
His; (19) Met; (20) Pro; (21) Arg; (22) Asp; (23) Cys; (24) Hcy; (25) GSH; (26) Glucose; (27)
H2O2; (28) NaClO. Conditions: λex = 365 nm.
-
-
2-
Mn2+, Al3+, Pb2+), negetively charged species (Ac-, SO42-, NO3 , PO4
,
S2O32-, CO32-, 0.01 mM for SO42-, PO42-, S2O32-, CO32-, 0.02 mM for Ac-,
NO3 ), amino acids (Phe, His, Met, Pro, Arg, Asp, Cys, Hcy, 0.01 mM
-
for each), GSH (0.01 mM), Glucose (0.01 mM), as well as reactive
oxygen species (H2O2, 0.01 mM; NaClO, 0.01 mM).
Visualizing semipermeability of cell membrane
The ratiometric fluorescent response of dhBBR towards pH and the
pharmacological properties of dhBBR33,34 inspired us to further
explore the potential of dhBBR to be utilized as a probe for ion
trapping. Before such an exploration, the biocompatibility of dhBBR
towards various cell lines was investigated first. As shown in Fig. S7,
dhBBR imposes little toxicity towards different cell types at a
concentration of 1 μM (with more than 80% cells alive), indicating
the feasibility of cell imaging study. For cell imaging, being different
from the reported pH-responsive probes which usually used nigericin
and monensin for intracellular pH calibration,35-38 we don’t use any
chemicals to calibrate intracellular pH because we want intracellular
Fig. 4 CLSM images of A549 cells (A) and HEK 293T cells (C) stained with dhBBR (1μM) in different pH PBS buffers for 30 min. (B, D) Relative PL intensity of dhBBR treated A549 cells
(B) and HEK 293T cells (D). Scale bar = 10 μm.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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DOI: 10.1039/D0SC02097D
Fig. 5 (A) Photostability of dhBBR and Curcumin under continuous scanning at 405 nm. I0 is the initial PL intensity, while I is that of the corresponding sample after a designated time
of irradiation. (B, C) CLSM images of HeLa cells stained with (B) dhBBR (1 μM) and (C) Curcumin (1 μM) before and after 200s of light irradiation. All the images share the same scale
bar: 10 μm.
(2H); HRMS (MALDI-TOF, m/z): [M]+ calcd. for C20H19NO4, 337.1314;
found, 337.1320.
Conclusions
In summary, dihydro berberine (dhBBR) was found to be an AIEgen.
Spectral measurement
Single crystal analysis, viscosity effect, as well as low-temperature
effect revealed that the AIE phenomenon of dhBBR originates from
the restriction of intramolecular vibration (RIV). Moreover, dhBBR
can serve as a fluorescent probe for visualizing ion trapping thanks
to its ratiometric fluorescent responses to pH and superior anti-
photobleaching ability.
The absorption and emission spectra were measured in PBS buffer
solutions (10 mM). A stock solution of dhBBR (1 mM) was prepared
in DMSO and was subsequently diluted to prepare 10 μM solutions
of dhBBR in PBS buffers with various pH (1, 2, 3, 4, 5, 6, 7, 7.4, 8, 9,
10, 11, 12). PBS buffers (10 mM) with varied pH were prepared by
using NaOH (1.0 M) or HCl (1.0 M) to adjust the pH. For the
calibration curve, 20 μL of stock solutions of dhBBR (1 mM) were
mixed with 980 μL different pH of PBS buffers in a quartz optical cell
with 1.0-cm optical path length at 25 °C, and spectral data were
recorded immediately. Excitation was at 365 nm and emission was
detected at 454 nm and 511 nm.
Experimental procedures
Materials and instrumentation
Chemicals for synthesis were purchased from Sigma-Aldrich or
Meryer used as received without any further purification. Dihydro
Berberine was prepared according to the reported literature.39 1H-
NMR spectrum was carried out on a Bruker AV 400 spectrometer.
High resolution mass spectra (HRMS) were recorded on a GCT
premier CAB048 mass spectrometer operating in MALDI-TOF mode.
Ultraviolet–visible (UV-vis) absorption spectra were taken on a
PerkinElmer Lambda 25 UV-Vis absorption spectrophotometer.
Photoluminescence spectra were recorded on a PerkinElmer LS 55
fluorescence spectrometer. The absolute fluorescence quantum
yields were measured on a Hamamatsu Absolute Quantum Yield
Spectrometer C11347.
Cell culturing
HeLa cells, A549 cells, and HEK 293T cells were purchased from
ATCC. All cells were cultured in Dulbecco's Modified Eagle's
Medium with 1% penicillin-streptomycin and 10% FBS, at 37 oC
in a humidified incubator with 5% CO2. The culture medium was
replaced every second day. By treating with 0.25% trypsin-EDTA
solution, the cells were collected after they reached confluence.
Cytotoxicity assay
HeLa cells, A549 cells, and HEK 293T cells were seeded in 96-well
plates at a density of 5000 cells per well, respectively. After 24 h cell
culture, various concentrations of dhBBR were added into the 96-
well plate. After another 24 h cell culture, the medium was removed
and the freshly prepared MTT medium solution (0.5 mg mL1, 100 μL)
was added into the 96-well plate. After incubation at 37 °C, 5% CO2
for 6 h, the MTT medium solution was removed carefully. After that, 100
μL DMSO was added into each well and the plate was gently shaken
at room temperature to dissolve all the formed precipitates. A
microplate reader was utilized to measure the absorbance at 570 nm
from which the cell viability could be determined. Cell viability was
Synthesis of dihydro berberine (dhBBR)
Anhydrous Berberine hydrochloride (1 mmol, 371.8 mg) and sodium
borohydride (1 mmol, 37.8 mg) were dissolved in pyridine, and then
the mixture was stirring slowly at room temperature. After that, ice
water was added into the system. The obtained light yellow powdery
solid was filtered, dried in vacuo to afford pure dhBBR (290.1 mg,
86%). 1H NMR (400 MHz, CDCl3, 25 °C), δ (ppm): 7.14 (1H), 6.71 (2H),
6.55 (1H), 5.92 (3H), 4.29 (2H), 3.82 (6H), 3.12-3.09 (2H), 2.87-2.84
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14 S. Wang, H. Yin, Y. Huang and X. Guan, Anal. Chem., 2018, 90,
expressed by the ratio of absorbance of the cells incubated with
dhBBR solution to that of the cells incubated with culture medium
only.
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8170–8177.
DOI: 10.1039/D0SC02097D
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Cell imaging
Cells were grown in a 35 mm Petri dish with a cover slip at 37
°C, 5% CO2. Firstly, Cells were incubated with different pH buffers
containing dhBBR (0.5, 1, 10 μM) or curcumin (10 μM) for 30 min
at 37 °C, 5% CO2. Then, the staining solution was removed and the
cells were washed with PBS of the same pH with the staining
solution for three times. After that, the cells were imaged using a
confocal microscopy (Zeiss laser scanning confocal microscope
LSM7 DUO). For dhBBR, the excitation wavelength was 405 nm,
and the emission filter was 400460 nm and 500-600 nm,
respectively; for Curcumin, the excitation wavelength was 405
nm, and the emission filter was 500600 nm.40
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Photobleaching assay
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HeLa cells stained with dhBBR were irradiated by 405 nm laser for
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was partially supported by the National Natural Science
Foundation of China (21788102), the Research Grants Council of
Hong Kong (16305518, N-HKUST609/19, A-HKUST605/16, and
C6009-17G), the Innovation and Technology Commission (ITC-
CNERC14SC01) and the Shenzhen Science and Technology Program
(JCYJ20180507183832774). We also thank the technical support
from AIEgen Biotech Co., Ltd.
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Chemical Science
Page 6 of 6
View Article Online
DOI: 10.1039/D0SC02097D
Abstract: In clinical chemotherapy, some basic drugs cannot enter the hydrophobic cell membrane because of
ionization in acidic tumor microenvironment, a phenomenon known as ion trapping. In this study, we developed a
method to visualize this ion trapping phenomenon by utilizing a pH-responsive ratiometric AIEgen, dihydro
berberine (dhBBR). By observing the intracellular fluorescence of dhBBR, we found that non-ionized dhBBR can
enter cells easier than ionized forms, which is in accordance with the concept of ion trapping. In addition, dhBBR
shows superior anti-photobleaching ability than Curcumin thanks to its AIE property. These results suggest that
dhBBR can serve as a bioprobe for ion trapping.