A fluorescent molecular rotor probe for tracking plasma membranes and exosomes in living cells

Article abstract of DOI:10.1039/d0cc03069d

A rotor-based probe MRMP-1 was designed and synthesized. MRMP-1 can bind to plasma membranes very quickly and stably with remarkable fluorescence enhancement. It can be used to monitor the dynamic changes in cell membranes in real-time under stimuli conditions. Importantly, MRMP-1 is the first rotor-based fluorescent sensor to label exosomes in living cells.

Full text of DOI:10.1039/d0cc03069d

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DOI: 10.1039/D0CC03069D
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A fluorescent molecular rotor probe for tracking plasma
membranes and exosomes in living cells
abd†
bd†
cd
cd
bd
Ling Feng,
Yusheng Xie, * Sung King Au-Yeung, Hagos Birhanu Hailu, Zhiyang Liu, Qingxin
Received 00th January 20xx,
Accepted 00th January 20xx
bd
bd
a
cd
cd
cd
bd
Chen, Jie Zhang, Qiuxiang Pang, Xi Yao, Mengsu Yang, Liang Zhang,* Hongyan Sun*
DOI: 10.1039/x0xx00000x
A rotor-based probe MRMP-1 was designed and synthesized. because an organic dye is relatively small and it might cause less
MRMP-1 can bind to the plasma membranes very fast but stably disruption for biomolecular targets, comparing with
with remarkable fluorescence enhancement. It can be used to nanoparticles and fluorescent proteins. Currently, the
monitor the dynamic changes of cell membranes in real-time under developed small molecular fluorescent probes mostly utilized
stimuli conditions. Importantly, MRMP-1 is the first rotor-based traditional and planar fluorophore. Taking the commercial dye
fluorescent sensor to label exosomes in living cells.
DiIs as example, DiIs are not preferably applied to track dynamic
change of plasma membranes because it would quickly enter
The plasma membrane, composed of phospholipids and
proteins, determines information and substances exchange
between the cell and its environment. Dysregulation of
cytoplasmic membrane-related events, such as cellular uptake,
membrane remodelling, signal transduction etc., have been
9a, 10
cells via lateral diffusion within short time.
Fluorescent
molecular rotors (FMRs) are a class of luminogen sensitive to
11
microenvironment such as viscoelasticity. Typically, they are
highly fluorescent in higher viscosity environment because of
the dominant competition of fluorescence against
intramolecular rotation. The molecule rotor can provide a
target-selective fluorescence signal with a high signal-to-noise
1
linked with a number of disease. For example, the cell
membrane viscoelasticity is believed to regulate protein-
2
protein interactions, and contribute to the development of
12
(S/N) ratio when they interact with biomolecular targets.
3
4
diabetes, atherosclerosis and Alzheimer’s disease. Another
example is that the rupture of cell membrane is the pivotal
Hence, the molecular rotor has the advantage of high
spatiotemporal imaging resolution. The plasma membrane has
viscoelastic feature that changes in various states, however, the
fluorescent molecular rotor probes for tracking plasma
5
evidence for apoptosis caused by drug. Thus, development of
chemical probes for real time imaging the plasma membrane
with high specificity and tracking the dynamic changes in detail
is significant for cell biology and pharmacology.
Fluorescence imaging is effective to identify cellular structures
and dissect the molecular mechanism of their functions. There
9b, 9c, 9e
membranes are rare.
Therefore, it is still essential to
develop the molecule rotor probe for plasma membrane
imaging with high specificity and excellent photophysical
characteristics.
6
have been a few fluorescent markers available for membrane.
They can be divided into three different families: aggregated
In this work, we developed a novel amphiphilic fluorescent
molecular rotor plasma membrane probe, MRMP-1, as shown
in Figure 1. It is composed of three key parts. The molecular
rotor, 9-(2-carboxy-2-cyanovinyl) julolidine (CCVJ) were utilized
because of its excellent increased fluorescence intensity based
on twisted intramolecular charge transfer (TICT) mechanism in
1
b, 7
8
nanostructures,
fluorescent proteins, and small molecule
The latter has attracted increasing attention
luminogens.5
d, 9
1
1c, 13
a
higher viscoelastic condition and its wide application.
The
Cancer and Aging Research Institution, School of Life Science, Shandong
University of Technology.
lipophilic palmitoyl group employed allows probe embedded
into cell membranes. To increase water-solubility, prevent from
endocytosis of probe, and enhance plasma membrane target
selectivity, a positively charged short peptide RRRR was further
introduced. MRMP-1 binds to the plasma membranes very fast
but stably with remarkable fluorescence enhancement. It also
can be used for long-term real-time tracking with good
b
Department of Chemistry and COSDAF (Centre of Super-Diamond and Advanced
Films), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong,
China, Email: yushengxie@outlook.com; hongysun@cityu.edu.hk
c
Department of Biomedical Sciences, College of Veterinary Medicine and Life
Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong,
China, Email: liangzhang.28@cityu.edu.hk
d
Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen
Research Institute of City University of Hong Kong, Shenzhen, 518057, P. R. China;
†
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any supplementary photostability. In addition, the dynamic change of the plasma
information available should be included here]. See DOI: 10.1039/x0xx00000x
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membrane caused by apoptosis induced by hydrogen peroxide indicated that MRMP-1 can bind to the plasma membranes
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DOI: 10.1039/D0CC03069D
were studied by MRMP-1. Particularly, employment of MRMP- specifically.
1
is capable of tracking the exosomes in cells.
To dissect the molecular mechanism of fluorescence
To synthesize MRMP-1, a synthesis route was illustrated in enhancement of MRMP-1 when binding to the plasma
Figure 1B. The synthesis commenced with rink-amide resin. By membrane, we first investigated the emission spectra of the
standard solid-phase peptide synthesis, a short peptide probe MRMP-1 in various solvents with different viscosities. In
K
mttRRRR was introduced on the resin. Selective deprotection of contrast with the negligible fluorescence in solvents with low
-Mtt at ε-amino group of lysine side chain by 2% TFA in DCM, viscosities such as water, PBS, methanol or acetonitrile, a very
and following coupling with palmitic acid afforded the palmityl strong emission with a maximum wavelength at 505 nm can be
modified Fmoc-KpalmRRRR-resin. Finally, by deprotection of observed obviously in the highly viscous glycerol solution (Fig.
terminal -Fmoc, further introduction of CCVJ, final cleavage by 2A). In the medium viscosity solvent DMSO, the fluorescence
9
5% TFA cocktail from resin, and purification by preparative can be detected as well. These results are consistent with the
HPLC, the final compound MRMP-1 was obtained and its features of fluorescent molecular rotor CCVJ, which shows
structure was determined by NMR and mass spectrometry.
higher fluorescence in a more restricted environment. We
further measured the fluorescence lifetime and quantum yield
of MRMP-1 in different concentration of glycerol solutions
(Table S1). The results indicated that the quantum yield and
fluorescence lifetime increased in more viscous glycerol
solution. The radiative and non-radiative decay constants were
determined from the experimentally measured quantum yields
and fluorescence lifetimes. They were summarized in Table S1
(Supporting Information). The results suggested that the non-
radiative decay constant increased in less viscous glycerol
solution. These data together proved the turn-on fluorescence
of MRMP-1 is based on twisted intramolecular charge transfer
(TICT) mechanism.
Figure 1. (A) Molecular structure of the plasma membrane probe
MRMP-1; (B) Synthetic scheme of MRMP-1. (C) CLSM image of MRMP-
1
(green, λex = 440 nm). (D) CLSM image of CellMask Deep Red (red,
Figure 2. (A) Fluorescence spectra of MRMP-1 (5 µM) in different types
of solvents. (B) Fluorescence spectra of MRMP-1 embedding into
liposomes, where the concentration of MRMP-1 ranged from 1 µM to
λex = 650 nm). (E) Merged image of panels C and panels D; CLSM stands
for confocal laser scanning microscopy. Living MCF-7 cells were
incubated with 20 µM MRMP-1 for 20 min or 10 µM CellMask Deep
Red for 10 min at 37 °C. Scale bar: 15.1 µm.
1
00 µM.
To further validate that MRMP-1 could insert into the
amphiphilic lipid bilayer, we prepared the liposomes and
performed the fluorescent properties studies with probe
MRMP-1. The liposomes with a diameter of 307.6 nm were
prepared by mixing hydrogenated phosphatidylcholine (hsPC)
and cholesterol (Fig.S1). The cholesterol was employed to
stabilize the liposomes. The liposomes were then fabricated by
incubating with MRMP-1 of various concentrations (1-100 μM)
for 20 min to obtain the probe-loaded liposomes (MRMP-
To test the design of probe as a plasma membrane tracker, we
next performed a proof of concept study. MRMP-1 (20 μM) was
incubated with MCF-7 cells. After 20 mins incubation, bright
fluorescence around the cell membrane was visible obviously
(Fig. 1C) using confocal laser scanning microscopy (CLSM). No
fluorescent signal was observed in the nucleus, cytoplasm and
other organelles. To further identify the fluorescent region, a
commercial cell membrane staining dye (CellMask Deep Red;
Thermo, H32721; Fig. 1D) was utilized to label cell membrane
specifically. By merging the imaged region from the two
channels, the fluorescence signal of MRMP-1 was found to
colocalize with the commercial membrane tracker signal,
unambiguously confirming that the MRMP-1 colocalized with
the plasma membrane (Fig. 1E). We further applied MRMP-1 to
label other cell lines, including LO2 and Hela cells (Fig. S3). It also
shows high specificity labeling of cell membranes. These results
1
@liposomes). The fluorescence of MRMP-1@liposomes was
enhanced dramatically as the concentration of MRMP-1
increased (Fig. 2B). On the contrary, the emission of MRMP-1
(50 μM) in water was rather weak (Fig. S2). These results
indicated that probe MRMP-1 inserted into the liposomes and
the intramolecular rotation of CCVJ was restricted by the lipid
bilayer structure. Combined the results of photochemical
properties of MRMP-1 in solution, they together clearly
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suggested that fluorescence increase of probe MRMP-1 upon LO
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cell membrane under H
O
2 2
stimuli conditions utilizing probe
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binding to cell membrane or its mimic (lipid bilayer structure) is MRMP-1. To do this, 50 mM hydrogen pDeOrIo: 1x0id.1e03w9/aDs0CaCd0d3e0d69tDo
due to twisted intramolecular charge transfer (TICT) induce cell apoptosis, and the timing processes were monitored
mechanism.
The use of higher concentration of probe or longer staining At beginning, the plasma membrane was highly fluorescent
times, but along with no or limited risk of penetrating through before H addition. However, the fluorescence intensity
with probe MRMP-1 (20 µM) using CLSM. As shown in Figure 3,
2 2
O
the cell membrane into the cytoplasm could endow this type of decreased at 10 minutes of the stimuli. And it disappeared
probe for continuous cell imaging of dynamic change events. To finally. These results were probably due to the rupture of cell
assess the potential of probe MRMP-1 as a real membrane membrane and the loss of the probe embedded in the
tracker, we next performed the dose- and time-dependent membrane. To eliminate that the diminished fluorescence is
fluorescence imaging studies. Firstly, the dose-effect of MRMP- caused by quenching of oxidized reagent hydrogen peroxide,
1
was investigated. MCF-7 cells were incubated with MRMP-1 we examined the emission spectra of probe MRMP-1 in
at various concentrations (5, 10 and 20 μM) for 20 min. The hydrogen peroxide solution. There is no difference in
fluorescence intensity of the cell membrane increased along fluorescence intensity of probe MRMP-1 between with and
with the increasing concentrations (Fig. S4A). In addition, the without H
distribution of fluorescence in the plasma membrane was confirmed the decreased fluorescence on cell membrane under
punctuate at μM probe concentration. When the stimuli conditions is attributed from rupture of cell
2 2
O experiments (Fig. S7). These data further
5
2 2
H O
concentration was higher (10 μM), the distribution of membrane.
fluorescence in the plasma membrane was almost uniform and
continuous. As the concentration of the probe was up to 20 μM,
the cell membrane was brightly labeled.
Next, we studied the effect of the staining time by incubating
MCF-7 cells with MRMP-1 at 20 μM for different timing (1, 5 and
2
0 min). As shown in Fig. S4B, with the incubating time
increasing, the fluorescent intensity enhanced. When incubated
for 20 min, the cell membrane was dramatically labeled by
MRMP-1. When we extended the incubation time to 2 h, the Figure 4. A) Immunoblots of the exosomes (anti-CD81) extracted from
migration of the probe into the cells was also observed (Fig. S5). mouse fibroblast L cells. Lane 1, 2, and 3 represent the exosome
These results suggested that MRMP-1 can be utilized as a bio- samples with no dilution, 1/2 dilution and 1/3 dilution respectively. B-
probe for tracking the cell membrane. Good photostability is D) CLSM images of MDA-MB-231 cells incubated with mouse fibroblast
vital for fluorescent probes’ biological applications. The L cells derived exosomes labeled by probe MRMP-1 (20 µM) and Dio (10
photostability of MRMP-1 and CellMask Deep Red in living cells µM) for various time (1, 2, 3 h). Green channel (MRMP-1 for exosome
was investigated by continuous laser exposure using CLSM. In labeling): λex = 440 nm, Blue channel (DAPI for nucleus staining): λex =
contrast with obvious diminishing of fluorescence intensity of 364 nm. Red channel (Dio for exosome labeling): λex = 488 nm. Scale
CellMask Deep Red, the probe MRMP-1 was still highly emissive, bar: 5 µm.
after continuous scanning for 9 min (Fig. S4C). These results
together suggested that probe MRMP-1 shows excellent Exosomes are membrane vesicles with diameters of 30-150 nm
photochemical properties and can be used a plasma tracker.
that are released by cells and present in biological fluids such as
1
5
blood and urine. They highly resemble liposomes in
membrane structure and size. Recent evidence suggested that
exosomes can deliver proteins, miRNA etc. to recipient cells,
thus mediating intercellular communication and affecting their
1
6
phenotypes. The innate ability of exosomes such as
biocompatibility, stability in blood circulation and capability to
avoid some of the pitfalls of liposomes has excited the drug
1
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delivery field. To study if exosomes could deliver the
functional cargos efficiently, tracking of exosomes in cells is very
necessary. The excellent performance of MRMP-1 in imaging
plasma membrane prompted us to investigate whether MRMP-
Figure 3. CLSM images of living LO
2 2 2
cells treated with 50 mM H O at
different timing utilizing probe MRMP-1 (20 µM). Scale bar: 30 µm.
1
could fluorescently label exosomes in cells. To the best our
knowledge, there is no rotor-based fluorescent probe applied
to track exosomes until now.
Having proven probe MRMP-1 as a membrane tracker, we tried
to investigate whether it can monitor morphology variation of
plasma membrane under stimuli conditions. Hydrogen peroxide
It was reported that fibroblast-derived exosomes can promote
breast cancer metastasis and motility via Wnt-planar cell
(H
2 2
O ) is reported to be a suitable apoptosis inducer and
1
8
polarity signalling. Thus, we chose mammary fibroblast
exosomes as our concept of study. At first, the exosomes were
isolated from the conditional media of mouse fibroblast L cells
eventually cause cell membrane damage and programmed cell
1
4
death. Thus, we next investigated the morphology variation of
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DOI: 10.1039/D0CC03069D
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CD81, Fig. 4A) and characterized by nanoparticle tracking
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Fig. S8). Next, 300 µL of exosomes (2.2 × 10 particles) were
incubated with MRMP-1 (40 µM) for 15 mins at room
temperature. Following centrifugation and removal of free dye, (6) (a)H. C. Ishikawa-Ankerhold, R. Ankerhold and G. P. C. Drummen,
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time (1, 2, 3 h). Exosomes labeled with DiO, a commercial dye
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imaging study. As shown in Figure 4B-D, the exosomes could be
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signal of MRMP-1 was found to largely colocalize with DiO in
both cell periphery and close to the nucleus, indicating a similar
performance of MRMP-1 with DiO, a broadly used commercial
dye for exosome labeling. Furthermore, more colocalized spots
could be observed with increased incubation time, indicating
that exosomes are progressively uptake by cells.
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Conclusions
In summary, we have successfully designed a bio-probe MRMP-
1
which can be used to track cell membrane. The rotor-based
characteristic was confirmed by photophysical properties of
MRMP-1 which showed high fluorescent intensity in the high
viscosity environment. Besides, MRMP-1 can be utilized to track
cell membrane universally in different cell lines and was highly
photostable. It can also be applied to monitor the dynamic
changes of cell membranes under stimuli conditions. Last not in
least, it can track exosomes in real-time mode. This provides
expected usage for cell membranes and exosomes in
bioimaging studies.
(
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This work was supported by the Science Technology and
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Conflicts of interest
There are no conflicts to declare.
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The rotor-based fluorescent probe to label exosomes in living cells
1