Dual aggregation-induced emission for enhanced fluorescence sensing of furin activity in vitro and in living cells

Article abstract of DOI:10.1039/c6cc09106g

The aggregation-induced emission (AIE) effect has recently been widely applied for biomarker sensing. But developing “smart” strategies to effectively aggregate the AIE fluorogen and additionally enhance the fluorescence emission remain challenging. In th

Full text of DOI:10.1039/c6cc09106g

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DOI: 10.1039/C6CC09106G
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Dual aggregation-induced emission for enhanced fluorescence
sensing of furin activity in vitro and in living cells
Received 00th January 20xx,
Accepted 00th January 20xx
Xiaomei Liuand Gaolin Liang*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Aggregation-induced emission (AIE) effect recently has been ease of operation, and needlessness of expensive instrumentation.
widely applied for biomarker sensing. But developing “smart” BL almost does not have background signal but the inexistence of
strategies to effectively aggregate the AIE fluorogen and luciferase in mammalian cells limits the applications of BL in rodents.
additionally enhance the fluorescence emission remain Compared with FL “Turn-Off” probes, FL “Turn-On” probes are
challenging. In this work, by integrating a biocompatible superior in lower background signal and recently a FL “Turn-On”
condensation reaction with an AIE fluorogen, we rationally nanoprobe was successfully developed for furin detection in vitro
9
designed a “smart” dual AIE probe Ac-Arg-Val-Arg-Arg-Cys(StBu)- and in vivo.
Lys(TPE)-CBT (1) for enhanced fluorescence sensing furin activity
Most of the FL “Turn-On” probes use the fluorescence resonance
in vitro and in living cells. Compared with single AIE probe Ac-Arg- energy transfer (FRET) effect to turn “On” their FL. For instance,
Val-Arg-Arg-Lys(TPE)-OH (1-Ctrl) which also subjects to furin upon the cleavage of the linker by an enzyme, the quencher is
cleavage, fluorescence emissions of 1 were additionally enhanced separated from the fluorophore, thus turning “On” the FL of the
1
0-13
1.7 folds and 3.4 folds in vitro and in living cells, respectively. We probe.
However, the FL enhancement of a “Turn-On” probe
envision that, in the near future, our “smart” strategy of enzyme- sometimes was not as high as expected probably due to that, after
instructed dual AIE could be widely applied for sensing (or imaging) the leave of the quencher from the probe, solubility of the
enzyme activity in vitro and even in vivo with dramatically fluorophore in the solvent dramatically decreased which resulted in
1
4
enhanced sensitivity.
another aggregation-caused quenching (ACQ) of the fluorophore.
Unlike the ACQ fluorophores, aggregation-induced emission (AIE)
fluorogens such as tetraphenylethylene (TPE) are nonemissive in
dissolved state but strongly emissive in aggregate state, which is
Enzymes are known as a class of natural proteins by which most
biochemical processes are efficiently catalyzed in a controlled
manner and precise specificity. The enzyme furin is one kind of
1
1
5
attributed to the restriction of intramolecular rotations (RIR). To
trans-Golgi protein convertase playing important roles in many
date, a variety of AIE-based fluorogens have been developed for
2
1
6
diseases including Alzheimer’s, Ebola fever, and cancer. Specifically,
sensing the activities of various enzymes such as trypsin, alkaline
1
7
18,19
furin was reported overexpressing in several cancers including
carcinomas of the head and neck, nonsmall-cell lung carcinomas,
phosphatase, and caspase.
Generally, the “Turn-On” FL of
these AIE probes was resulted in the direct aggregation of the
fluorogens after enzyme cleavage (we call “Single AIE” in this work).
Nevertheless, to the best of our knowledge, there was no report of
further aggregation (we call “Dual AIE” in this work) of the AIE
fluorogens which would obviously result in enhanced FL “Turn-On”
for more efficient sensing (or imaging) of enzyme activity.
3
and glioblastomas. The peptide sequences Arg-X-Lys/Arg-Arg↓X (X
can be any amino acid residue and ↓ indicates the cleavage site)
4
were reported to be the preferential substrates for furin cleavage.
Based on the specific substrates, many probes have been
developed for sensing (or imaging) furin activity with different
5
6
modalities including fluorescence (FL), magnetic resonance,
Recently, Rao and Liang developed a biocompatible click
condensation reaction between the 1,2-aminothiol group of
cysteine (Cys) and the cyano group of 2-cyanobenzothiazole (CBT)
which can be controlled by pH, reduction, or protease for the self-
7
8
nuclear, bioluminescence (BL), etc. Among the techniques above,
FL and BL have attracted much attention due to their simplicity,
2
0,21
assembly (or aggregation) of nanoparticles.
By conjugation of an
imaging tag to the monomer, this CBT-Cys condensation reaction
was successfully employed to self-assemble (or aggregate)
nanoprobes to concentrate the signal for enhanced sensing (or
2
2-24
imaging) of enzyme activity in vitro and in vivo.
Unluckily, if the
tag was a conventional fluorophore, aggregation of nanoparticles
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by this condensation reaction would result in ACQ of the FL and
Journal Name
We began the study with the syntheses of 1, 1-Scr, and 1-
View Article Online
DOI: 10.1039/C6CC09106G
only subsequent disassembly of the nanoparticles could partially Ctrl. The syntheses are facile and straightforward (Scheme S1-
9
restore the FL for enzyme detection (or imaging).
S4 in the ESI†). Briefly, TPE-COOH was synthesized following
2
5
Inspired by above pioneering studies, in this work, we intended the literature.
Peptide sequence Ac-Arg-Val-Arg-Arg-
to integrate the AIE property of a TPE molecule with a furin- Cys(StBu)-Lys-OH (A) with protecting groups was synthesized
controlled CBT-Cys condensation to dually aggregate the TPE with solid phase peptide synthesis (SPPS). Coupling of A with
fluorogen. Consequently, the “Dual AIE” effect could be realized for CBT in the presence of isobutyl chloroformate yielded B after
enhanced FL sensing and imaging of furin activity. To achieve this, high-performance liquid chromatography (HPLC) purification.
as shown in Scheme 1, we rationally designed a probe Ac-Arg-Val- Deprotection of B yielded C and coupling C with TPE-NHS ester
Arg-Arg-Cys(StBu)-Lys(TPE)-CBT (1) which contains following yielded 1 after HPLC purification. Synthesis of 1-Scr was similar
components: an Ac-Arg-Val-Arg-Arg (RVRR) peptide sequence which to that of 1 except the peptide sequence was scrambled. 1-Ctrl
not only improves the cellular uptake of the probe but also is the was obtained by direct coupling of Ac-Arg-Val-Arg-Arg-Lys-OH
substrate for furin cleavage; a CBT motif and a disulfided Cys motif with TPE-NHS ester after HPLC purification. The compounds
1
13
for CBT-Cys condensation; and a TPE motif conjugating to the side were fully characterized with H NMR, C NMR, and mass
chain of a lysine (Lys) motif. As illustrated in Scheme 1, after 1 spectrometry (MS) analyses (Fig. S1-S11 in the ESI†). After
entering furin-overexpressing tumor cells (e.g., MDA-MB-468 cells), obtaining the pure compounds, we firstly investigated the AIE
its disulfide bond on the cysteine motifs is reduced by intracellular property of the compounds in DMSO/H
2
O mixtures. At DMSO
glutathione (GSH) and its RVRR peptide substrate is cleaved by furin fractions less than 50%, TPE-COOH exhibited obvious AIE
to yield the reactive intermediate 1-Cleaved. Theoretically, 1- property with a fluorescence emission peak at 470 nm (Fig.
Cleaved is more hydrophobic than 1 and conversion of 1 to 1- S12 in the ESI†). The transmittance spectra of 1 in
Cleaved will lead to the first aggregation of the TPE fluorogen (i.e., DMSO/water mixtures indicated that, at DMSO fractions more
single AIE). Instantly, the free 1,2-aminothiol group and the cyano than 5%, 1 was totally dissolved in the mixture (Fig. S13A in the
group on the CBT motif of 1-Cleaved condenses to yield the more ESI†). Fluorescence emission spectra indicated that, at DMSO
hydrophobic dimer (i.e., 1-Dimer) which self-assembles (or fraction of 5%, while TPE-COOH had strong emission at 470 nm,
aggregates) into the nanoparticles (i.e., 1-NPs) at (or near) the none of the compounds 1, 1-Scr, or 1-Ctrl showed any
locations of activated furin (i.e. Golgi bodies), as demonstrated emission at this wavelength (Fig. S13B in the ESI†). Thus, we
9
,23
previously.
Conversion of 1-Cleaved to 1-Dimer and 1-NPs will chose the aqueous solution containing 5% DMSO as the
lead to further aggregation of the TPE fluorogen (i.e., dual AIE) and solvent for the in vitro experiments below.
thus its fluorescence is additionally enhanced. With this dual AIE
strategy, furin activity in tumor cells could in turn be more
sensitively detected (or imaged). To validate our hypothesis, a
scrambled analog Ac-Arg-Lys(TPE)-Arg-Cys(StBu)-Arg-Val-CBT (1-Scr),
which is insusceptible to furin cleavage (i.e., no AIE effect), was
synthesized for parallel study (Fig. 1). As we mentioned above, 1-
Cleaved possesses single AIE effect but it is a reactive intermediate
and can hardly be captured. Thus, in this work, another control
probe Ac-Arg-Val-Arg-Arg-Lys(TPE)-OH (1-Ctrl), which only subjects
to furin cleavage but does not take condensation reaction,
possessing single AIE effect was also synthesized for the proof of
our concept (Fig. 1).
Fig. 1 Chemical structures of 1, 1-Scr, and 1-Ctrl. Schematic
illustration of 1-Ctrl for furin cleavage. The red arrows indicate
the furin cleavage sites.
In order to qualitatively and quantitatively measure the
enzymatic cleavage efficiency with HPLC, we used 100 μM of 1,
1-Scr, or 1-Ctrl for the enzymatic assay. Dissolved in furin
buffer containing 5% DMSO and 1 mM tris(2-carboxyethyl)-
phosphine (TCEP) at pH 7.4, none of 100 μM 1, 1-Scr, or 1-Ctrl
solution showed detectable fluorescence emission at 470 nm
(
Fig. 2A-2B and Fig. S14A in the ESI†). However, after the
-1
addition of 1 nmol U furin and 4 h incubation at 37 °C, we
observed the fluorescence of 1 was turned on and its emission
intensity at 470 nm increased approximately 109 folds
Scheme 1. Schematic illustration of furin-controlled dual
aggregation-induced emission for enhanced fluorescence sensing of
furin activity.
(
intensity: 699.9 vs 6.4) (Fig. 2A). Interestingly, as 1-Ctrl was
also subjected to furin cleavage, we observed its fluorescence
emission at 470 nm increased about 63 folds after furin
addition (intensity: 335.2 vs 5.3) (Fig. 2B). This was due to the
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single AIE effect of 1-Ctrl after furin cleavage. HPLC analysis and collected the peaks for matrix-assViisetweAdrticle lOansliener
indicated that, besides small amount of the reduction product desorption/ionization (MALDI) mass specDtOroI:s1c0o.1p0i3c9/aCn6aClyCs0e9s1.06AGs
of 1 by TCEP (i.e., 1-Red), only the condensation product 1- shown in Fig. 2D, a new peak at retention time of 31.5 min,
Dimer but not the active intermediate 1-Cleaved appeared on whose mass was identical to that of 1-Dimer (Fig. S20 in the
the HPLC trace (Fig. S15B in the ESI†), suggesting the ESI†), appeared in the HPLC trace of the reaction mixture of 1.
condensation was much faster than the aggregation of 1- This in principle testified our hypothesis of the furin-controlled
Cleaved. Therefore, we deduced that, compared with 1-Ctrl, dual AIE of 1 in Scheme 1. The enzymatic parameters Kcat and
-1
the 1.7-fold (109/63) additional emission enhancement at 470
m
Kcat/K of furin towards 1 were calculated to be 7.84 min and
-1
-1
nm of 1 was resulted from furin-controlled aggregation of 1- 0.047 μM min , respectively (Fig. S21 in the ESI†). HPLC trace
Cleaved followed by further aggregation of the condensation of the reaction mixture of 1-Ctrl showed a main peak at
production 1-Dimer (i.e., dual AIE). While 1-Scr was impervious retention time of 19.7 min (Fig. 2D), whose mass agreed with
to furin, its fluorescence emission at 470 nm did not show that of 1-Ctrl-Cleaved (Fig. S22 in the ESI†), suggesting the
obvious enhancement after furin addition, as expected (Fig. emission enhancement of 1-Ctrl was induced by furin-cleavage
S14A in the ESI†).
single AIE effect. HPLC trace of the mixture of 1-Scr incubated
UV-vis spectroscopy, dynamic light scattering (DLS), and with furin only showed the reduction product of 1-Scr by TCEP
transmission electron microscope (TEM) were used to verify in the furin buffer (i.e., 1-Scr-Red) (Fig. S14B in the ESI†),
the formation and characterize the aggregates obtained. The echoing above that 1-Scr did not possess AIE effect.
increased UV-vis absorptions from 500 nm to 700 nm (as a
After in vitro investigation of the specific dual AIE
result of light scattering) of 1 and 1-Ctrl after incubation with characteristics of 1, we then applied 1 for enhanced
furin revealed the formation of aggregates in their respective fluorescence imaging of furin activity in living cells. Different
solutions (Fig. S16 in the ESI†), which indicated above emission from the in vitro studies, we used 5 μM of the probes for the
enhancement of 1 and 1-Ctrl were resulted from their cell imaging experiments. Before that, cytotoxicity of 1, 1-Scr,
respective AIE effect. DLS measurements showed the average and 1-Ctrl on furin-overexpressing MDA-MB-468 cells were
hydrous dynamic diameters of the aggregates of 1 (i.e., 1-NPs) investigated.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
and 1-Ctrl (i.e., 1-Ctrl-NPs) were about 190.1 nm and 138.7 nm, trazolium bromide (MTT) assay indicated that, after being
respectively (Fig. S17 and S18 in the ESI†). TEM image of 1-NPs incubated with 1, 1-Scr, or 1-Ctrl at 37 °C, more than 90% of
indicated that the aggregates have an average diameter of the cells survived up to 8 hour at compound concentration up
95.0 ± 5.6 nm (Fig. 2C and Fig. S19 in the ESI†).
to 40 μM, respectively (Fig. S23 in the ESI†), suggesting
compound concentration of 5 μM is safe for cell imaging.
Similarly, minimum DMSO concentration in Dulbecco’s
Modified Eagle Medium (DMEM) to ensure a total solubility of
1
in the medium was tested before the probe being applied for
cell imaging. Transmittance spectra of 1 in DMEM containing
different concentrations of DMSO indicated that 1% was the
minimum DMSO concentration for 1 to be totally dissolved (Fig.
S24 in the ESI†). To overcome the interference from
intracellular Cys and warrant the condensation of 1 for the
2
3
aggregation of 1-NPs inside cells, we used 50 μM C (i.e., Ac-
Arg-Val-Arg-Arg-Cys(StBu)-Lys-CBT, see Scheme S2) to co-
incubate with 5 μM 1 (or 1-Scr, 1-Ctrl as well) for the cell
imaging experiments. Time course cell imaging indicated that,
while 5 μM 1 was nonfluorescent in the culture media (t = 0
min), fluorescence of the cells gradually turned on and reached
to its intensity plateaus after 60 min (Fig. S25 in the ESI†),
suggesting that the uptaken 1 was totally converted to 1-NPs
by intracellular furin. We thus chose incubation time of 60 min
for the following comparative cell imaging studies.
Fig. 2 (A) Fluorescence spectra of 100 μM 1 (black), 100 μM 1
-1
incubated with 1 nmol U furin at 37 °C for 4 h in furin buffer
red). (B) Fluorescence spectra of 100 μM 1-Ctrl (black), 100
(
-1
μM 1-Ctrl incubated 1 nmol U furin at 37 °C for 4 h in furin
After the MDA-MB-468 cells were incubated with 5 μM 1, 1-
TEM image of 1-NPs. (D) HPLC trace of 100 μM 1 (black), the Scr, or 1-Ctrl, together with 50 μM C for 60 min at 37 °C, the
mixture of 100 μM 1 incubated with 1 nmol U furin at 37 °C cells were washed with PBS for three times prior to imaging,
for 4 h (blue), 100 μM 1-Ctrl (red), 1-Ctrl incubated with 1 respectively. As shown in Fig. 3 (statistic of the FL intensity was
buffer (red), respectively. Excitation wavelength: 320 nm. (C)
-1
-1
nmol U furin at 37 °C for 4 h (green).
shown in Fig. S26 in the ESI†), the cells treated with 1 had the
highest FL emission intensity (mean intensity: 79.6 ± 16.9),
while the emission of those cells treated with 1-Ctrl was much
lower (mean intensity: 23.4 ± 4.6) and those cells treated with
To chemically validate that above dual AIE effects of 1 was
induced by furin-controlled condensation while the single AIE
effect of 1-Ctrl was induced by furin-cleavage, we directly
injected the above two incubation mixtures into a HPLC system
1-Scr had the lowest emission intensity (mean intensity: 3.8 ±
1.3). These results indicated that, compared with 1-Scr, single
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Journal Name
AIE effect of 1-Ctrl enhanced the FL of TPE fluorogen 6.2 folds The authors are grateful to Chuanzhi Yao for his assVisietwanAcrteicleoOf ntlhinee
23.4/3.8) for cell imaging, as expected. Nevertheless, our synthesis of TPE-COOH. This work was supDpOoIr:t1e0d.10b3y9/CCo6llCaCb0o9ra10ti6vGe
(
fantastic dual AIE probe 1 additionally enhanced the FL of Innovation Center of Suzhou Nano Science and Technology, Hefei
single AIE probe 1-Ctrl 3.4 folds (79.6/23.4) for the best cell Science Center CAS (2016HSC-IU010), Ministry of Science and
imaging. To verify that above strongest fluorescence intensity Technology of China (2016YFA0400904), and the National Natural
from 1-incubated cells was resulted from furin-controlled dual Science Foundation of China (Grants U1532144 and 21675145).
AIE effect as proposed in Scheme 1, we pretreated the cells
with a furin inhibitor II (H-(D)Arg-Arg-Arg-Arg-Arg-Arg-NH
5
5
inhibitor-treated cells dramatically decreased (Fig. S27 in the
ESI†). Collectively, above results suggest that our dual AIE
probe 1 can be used for enhanced FL sensing of furin activity in
living cells.
2
) at
00 μM for 30 min before incubating the cells with 5 μM 1 and
0 μM C. Compared with 1-treated cells, FL signals of the
Notes and references
1
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Fig. 3 Differential interference contrast images (top row),
fluorescence images (middle row, DAPI channel), and merged
images (bottom row) of MDA-MB-468 cells incubated with 5
1
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Conclusions
In conclusion, by integrating a biocompatible condensation
reaction with the AIE property of a TPE fluorogen, we
rationally designed a “smart” probe 1 for furin-controlled dual
aggregation of the TPE molecule and applied 1 for enhanced
fluorescence sensing of furin activity in vitro and in living cells.
In vitro results indicated that, compared with single AIE probe
2
2
2
1-Ctrl, the dual AIE probe 1 additionally enhanced the FL
emission 1.7 folds. Living cell imaging results indicated that the 23 Y. Yuan, L. Wang, W. Du, Z. L. Ding, J. Zhang, T. Han, L. N. An, H.
F. Zhang and G. L. Liang, Angew. Chem., Int. Ed., 2015, 54, 9700.
4 Y. Yuan, H. B. Sun, S. C. Ge, M. J. Wang, H. X. Zhao, L. Wang, L. N.
An, J. Zhang, H. F. Zhang, B. Hu, J. F. Wang and G. L. Liang, ACS
Nano, 2015, 9, 761.
5 G. D. Liang, J. W. Y. Lam, W. Qin, J. Li, N. Xie and B. Z. Tang,
Chem. Commun., 2014, 50, 1725.
FL emission of 1-treated cells increased 3.4 folds compared
with that of cells treated with single AIE probe 1-Ctrl. We
envision that, in the near future, our “smart” strategy of
enzyme-instructed dual AIE could be widely applied for sensing
2
2
(or imaging) enzyme activity in vitro and even in vivo with
dramatically enhanced sensitivity.
Acknowledgements
4
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