Peptide-Induced AIEgen Self-Assembly: A New Strategy to Realize

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Peptide-Induced AIEgen Self-Assembly: A New Strategy to Realize Highly Sensitive Fluorescent Light-Up Probes Aitian Han, Huaimin Wang, Ryan Tsz Kin Kwok, Shenglu Ji, Jun Li, Deling Kong, Ben Zhong Tang, Bin Liu, Zhimou Yang, and Dan Ding Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00023 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 12, 2016

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Analytical Chemistry

Peptide-Induced AIEgen Self-Assembly: A New Strategy to Realize Highly Sensitive Fluorescent Light-Up Probes Aitian Han,†,# Huaimin Wang,†,# Ryan T. K. Kwok,‡ Shenglu Ji,† Jun Li,† Deling Kong,† Ben Zhong Tang,‡ Bin Liu,*,§ Zhimou Yang,*,† and Dan Ding*,†,§ †

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China. Tel: +86-22-23501229, Fax: +86-22-23498775, E-mail: [email protected] (D. Ding); Tel: +86-22-23502875, Fax: +86-22-23498775, [email protected] (Z. Yang) ‡ Department of Chemistry, Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) § Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585. Tel: +65-65168049, Fax: +65-67791936, E-mail: [email protected] (B. Liu) ABSTRACT: Fluorescent light-up probes with aggregation-induced emission (AIE) characteristics have recently attracted great research interest due to their intelligent fluorescence activation mechanism and excellent photobleaching resistance. In this work, we report a new, simple and generic strategy to design and prepare highly sensitive AIE fluorescent light-up bioprobe through facile incorporation of a self-assembling peptide sequence GFFY between the recognition element and the AIE luminogen (AIEgen). After the bioprobes respond to the targets, the peptide GFFY is capable of inducing the ordered self-assembly of AIEgens, yielding close and tight intermolecular steric interactions to restrict the intramolecular motions of AIEgens for excellent signal output. Using two proof-of-concepts, we have demonstrated that self-assembling peptide-incorporating AIE light-up probes show much higher sensitivity in sensing the corresponding targets in both solutions and cancer cells, as compared to those without GFFY induced selfassembly. Taking the probe TPE-GFFYK(DVEDEE-Ac) for example, a detection limit as low as 0.54 pM can be achieved for TPEGFFYK(DVEDEE-Ac) in caspase-3 detection, which is much lower than that of TPE-K(DVED-Ac) (3.50 pM). This study may inspire new insights into the design of advanced fluorescent molecular probes.

The emergence of molecular fluorescent light-up probes that can specifically turn on their fluorescence in the presence of targets has opened up a new opportunity to advance biosensing and bioimaging.1-5 As compared to the conventional fluorescent molecular probes, the ones with fluorescence light-up characteristics are more intelligent and hold the advantages of less false positive signals and larger target-to-background ratios.6-8 To date, several strategies have been explored for designing molecular fluorescent light-up probes based on various fluorescence quenching/activation mechanisms including fluorescence resonance energy transfer (FRET),9,10 intramolecular spirocyclization,11 photoinduced electron transfer (PeT),12 excited-state intramolecular proton transfer (ESIPT),13 and intramolecular charge transfer (ICT), etc..14-17 Although there have been plenty of successful studies on molecular fluorescent light-up probes for biosensing and bioimaging, many of these probes suffer from the limitations such as low fluorescence turn-on ratios, inability to be used in living organisms, or poor photobleaching thresholds. Recently, organic luminogens with aggregation-induced emission (AIEgen) have been developed,18-20 which have shown excellent performance in fluorescent light-up sensing and imaging with high signal-to-noise ratio, facile operation in living organisms, low cytotoxicity and strong photobleaching resistance.21-24 The AIEgens are often propeller-shaped and

possess rotating units. As the molecular motions of rotating units result in fast non-radiative decay of the excited states, the AIEgens are non-fluorescent as molecular species. Nevertheless, in aggregate state, the AIEgens emit brightly due to the well-known restriction of intramolecular motion (RIM) mechanism.25,26 This fluorescence quenching/activation mechanism makes AIEgens an ideal fluorescent material to construct fluorescent light-up bioprobes, as the interactions between the probes and bioactive molecules (e.g., enzymes, non-enzymatic proteins, etc.) in biological environments often result in molecular aggregation and the occurrence of RIM.27-29 For instance, by employing an iconic AIEgen, tetraphenylethene (TPE), we developed a bioprobe of TPE-K(DVED-Ac) for sensing caspase-3, a protease playing key roles in mediating cell apoptosis. TPE-K(DVED-Ac) was non-emissive in aqueous media thanks to the hydrophilic peptide sequence of DEVD, which endows great water-solubility of the probe. In the presence of caspase-3, the hydrophilic DEVD was cleaved by the enzyme and the hydrophobic TPE-K residues formed nanoaggregates, which restricted the intramolecular rotations of phenyl rings in TPE and thus significantly activated the TPE fluorescence.30 A holy grail in fluorescent molecular probes is to detect and visualize analytes with extremely high sensitivity.31-33 Hence, more sensitive fluorescent light-up probes with larger fluores-

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cence turn-on ratios are in perpetual pursuit. In this contribution, we aim to develop a new strategy to fabricate highly sensitive AIE light-up bioprobe by employing a short selfassembling peptide sequence GFFY. After responding to the targets, the peptide GFFY is able to induce the ordered selfassembly of AIEgens, which would terrifically restrict the intramolecular motions of AIEgens, rendering the probes with high sensitivity. This study thus offers a simple and effective strategy to prepare highly sensitive AIE fluorescent light-up probes.

EXPERIMENTAL SECTION Materials. Fmoc-OSu and other Fmoc-amino acids were purchased from GL Biochem (Shanghai, China). 2-Cl-trityl chloride resin (1.0-1.2 mmol/g) was obtained from Nankai University Resin Co. Ltd. Bovine serum albumin (BSA), lysozyme, trypsin, pepsin, alkaline phosphatase, proteinase K, cathepsin B, papsin, piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES), penicillin-streptomycin solution, trypsinethylenediaminetetraacetic acid (EDTA) solution and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Recombinant human caspase-3 was customized from R&D Systems. Caspase3 inhibitor 5-[(S)-(+)-2(methoxymethyl)pyrrolidino]sulfonylisatin was purchased by Calbiochem. Staurosporine (STS) was provided by Biovision. Chemical reagents and solvents were used as received from commercial sources. 1-[4-(Isothiocyanatomethyl)phenyl]1,2,2-triphenylethene (TPE-ITC) was synthesized according to the previous report.44 Characterization. 1H NMR spectra were recorded on a Bruker ARX 400. HPLC was carried out at a LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.1% of TFA) and water (0.1% of TFA) as the eluents. LCMS was conducted at the LCMS-20AD (Shimadzu) system. UV-vis absorption spectra and Photoluminescence (PL) spectra were measured on a Shimadzu UV-1700 spectrometer and a Perkin-Elmer LS 55 spectrofluorometer, respectively. Transmission electron microscopy (JEM-2010F, JEOL, Japan) was employed to study the sample morphology. Synthesis of TPE-GFFYK(DVEDEE-Ac). Syntheses of Ac-E(OtBU)E(OtBU)D(OtBU)E(OtBU)VD(OtBU)-COOH and Fmoc-GFFYK were described in the SI. To a solution of Ac-E(OtBU)E(OtBU)D(OtBU)E(OtBU)VD(OtBU)-COOH (50 mg, 47.3 µmol) in DCM (10 mL) was added Nhydroxysuccinimide (NHS) (6 mg, 52 µmol) and N,N’dicyclohexylcarbodiimide (DCC) (11.5 mg, 56.5 µmol). After being stirred for 3 h at room temperature, the precipitation was filtered and the supernatant was evaporated to dryness in vacuo. The resulting product was then dissolved in 5 mL of DMF, followed by adding a DMF solution of Fmoc-GFFYK (62.65 mg, 71 µmol). The pH value of the mixture was adjusted to around 8.0 using N,N-diisopropylethylamine (DIPEA). After reaction at room temperature for 12 h, the mixture was purified by HPLC to yield FmocGFFYK(D(OtBU)VE(OtBU)D(OtBU)E(OtBU)E(OtBU)-Ac) (53.6 mg, 80% yield). Subsequently, the product was cleaved using 95% of TFA with 2.5% of trimethylsilane (TMS) and 2.5% of H2O for 30 min. After the solution was concentrated by the rotary evaporator, the piperidine (20%) in anhydrous DMF was used to cleave the Fmoc group for 30 min. The obtained product was purified by HPLC to yield NH2-

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GFFYK(DVEDEE-Ac) in 70% yield. To prepare TPEGFFYK(DVEDEE-Ac), NH2-GFFYK(DVEDEE-Ac) (26.4 mg, 18.6 µmol) was added to a solution of TPE-ITC (5 mg, 12.4 µmol) in dimethyl sulfoxide (DMSO). DIPEA was used to adjust the final pH of the mixture to 8-9. After reaction for 24 h at room temperature, the final product was purified by HPLC to yield TPE-GFFYK(DVEDEE-Ac) in 70% yield. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 7.40-7.47 (m, 1H), 6.857.25 (m, 28H), 6.60-6.67 (d, 2H), 4.41-4.62 (m, 7H), 3.98-4.33 (m, 7H), 3.87-3.95 (m, 1H), 3.54-3.59 (m, 2H), 3.15-3.19 (d, 1H), 2.87-3.08 (m, 6H), 2.60-2.81 (m, 5H), 2.15-2.38 (m, 7H), 1.88-2.05 (m, 4H), 1.82-1.87 (m, 3H), 1.65-1.81 (m, 4H), 1.50-1.63 (m, 2H), 0.72-0.88 (t, 6H). MS: calcd M+ = 1822.98, obsvd 1/2(M + H)+ = 912.35. Synthesis of TPE-K(DVED-Ac). TPE-K(DVED-Ac) was synthesized following the same experimental procedures as that for the preparation of TPE-GFFYK(DVEDEE-Ac), using Fmoc-K and Ac-D(OtBU)E(OtBU)VD(OtBU)-COOH as the starting materials. The final product was purified by HPLC to yield TPE-K(DVED-Ac) in 70% yield. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 7.01-7.18 (m, 11H), 6.94-7.01 (m, 6H), 6.87-6.93 (d, 2H), 4.61-4.70 (m, 1H), 4.44-4.50 (m, 3H), 4.274.39 (m, 2H), 4.11-4.24 (m, 2H), 3.68-3.76 (m, 1H), 3.53-3.61 (m, 2H), 2.61-2.73 (m, 1H), 2.41-2.45 (m, 1H), 2.28-2.39 (m, 2H), 2.19-2.27 (m, 2H), 1.92-2.05 (m, 2H), 1.79-1.85 (m, 3H), 1.56-1.77 (m, 4H), 1.29-1.39 (m, 2H), 1.11-1.20 (m, 2H), 0.72-0.90 (m, 7H). MS: calcd M+ = 1049.42, obsvd (M + H)+ = 1050.45. Synthesis of TPE-GFFYE-SS-EE. Fmoc-cystamine succinic acid (Fmoc-SS) for solid phase peptide was first synthesized according to previous report.45 The peptide of NH2GFFYE-SS-EE was then synthesized by standard Fmoc SPPS using 2-chlorotrityl chloride resin as well as the corresponding N-Fmoc protected amino acids with side chains properly protected and Fmoc-SS. To prepare TPE-GFFYE-SS-EE, NH2GFFYE-SS-EE (21.5 mg, 18.6 µmol) was added to a solution of TPE-ITC (5 mg, 12.4 µmol) in dimethyl sulfoxide (DMSO). DIPEA was used to adjust the final pH of the mixture to 8-9. After reaction for 24 h at room temperature, the final product was purified by HPLC to yield TPE-GFFYE-SS-EE in 70% yield. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 8.12-8.18 (m, 2H), 7.98-8.05 (m, 3H), 7.07-7.24 (m, 22H), 6.92-7.05 (m, 12H), 6.61-6.67 (d, 2H), 4.41-4.50 (m, 2H), 4.15-4.28 (m, 4H), 3.17 (s, 1H), 2.86-3.02 (m, 4H), 2.70-2.80 (t, 7H), 2.652.69 (m, 2H), 2.20-2.39 (m, 11H), 1.87-2.01 (m, 3H), 1.641.85 (m, 3H), 1.19-1.31 (m, 5H). MS: calcd M+ = 1556.55, obsvd (M + H)+ = 1557.90. Synthesis of TPE-SS-EE. TPE-SS-EE was synthesized following the same experimental procedures as that for the preparation of TPE-GFFYE-SS-EE. The final product was purified by HPLC to yield TPE-SS-EE in 70% yield. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 7.07-7.17 (m, 9H), 6.90-7.06 (m, 10H), 4.48-4.61 (m, 2H), 4.23-4.31 (m, 1H), 4.12-4.21 (m, 1H), 3.56-3.61 (m, 3H), 3.17 (s, 1H), 2.83-2.92 (t, 2H), 2.702.80 (m, 3H), 2.54 (s, 5H), 2.17-2.42 (m, 8H), 1.61-2.08 (m, 5H). MS: calcd M+ = 913.28, obsvd (M + H)+ = 914.40. Apoptosis Imaging in Cancer Cells. HeLa cancer cells were cultured in confocal imaging chambers at 37 °C. Subsequently, TPE-GFFYK(DVEDEE-Ac) and TPE-K(DVED-Ac) in cell culture medium at a concentration of 0.2 µM were added to the chambers, respectively. After incubation at 37 oC for 2 h, the cells were washed and incubated with staurosporine


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(STS, 2 µM) in cell culture medium for 1 h to trigger cell apoptosis. The cells were then fixed with 4% paraformaldehyde and imaged by confocal laser scanning microscope (CLSM, Zeiss LSM 410, Jena, Germany) equipped with DAPI filter. Intracellular Thiol Level Imaging. After 80% confluence of A549 cancer cells in the chambers at 37 oC, 1 µM of TPEGFFYE-SS-EE and TPE-SS-EE in cell culture medium were added to the chambers and co-incubated with the cells for 2 h, respectively. Then the cells were washed and incubated for further 4 h at 37 oC. After that, the cells were fixed with 4% paraformaldehyde and imaged by CLSM equipped with DAPI filter.

RESULTS AND DISCUSSION Scheme 1. Schematic illustrations of (A) the TPE aggregates and orderly self-assembly of TPE-GFFY as well as (B) the random and orderly arrangements of butterflies in a box.

der, it is much more difficult for them to flap their wings in the box, as compared to the randomly arranged ones (Scheme 1B). To test our hypothesis, the probe of TPEGFFYK(DVEDEE-Ac) was designed and synthesized. Its chemical structure and synthetic route are shown in Scheme 2A and Scheme S1 in the Supporting Information (SI). After preparation of NH2- GFFYK(DVEDEE-Ac) via standard solid-phase 9-fluorenylmethoxycarbonyl peptide chemistry in combination with solution synthesis, the probe TPEGFFYK(DVEDEE-Ac) was obtained by the addition reaction between the N-terminal amine group of peptide and the isothiocyanate group on 1-[4-(Isothiocyanatomethyl)phenyl]-1,2,2triphenylethene (TPE-ITC). The 1H NMR and LC-MS spectra are depicted in Figures S1 and S2 in the SI, to characterize the probe. As a control, the previously reported TPE-K(DVEDAc)30 (Scheme 2A) without incorporation of self-assembling peptide GFFY was also synthesized for comparison according to the synthetic route in Scheme S2. TPE-K(DVED-Ac) was characterized with 1H NMR and LC-MS as well (Figures S3 and S4 in the SI). Scheme 2. Chemical structures of (A) TPEGFFYK(DVEDEE-Ac) and TPE-K(DVED-Ac) as well as (B) TPE-GFFYE-SS-EE and TPE-SS-EE. A OH

H N

H N

O N H

S

O

H N

N H

O

O

H N

OH

O O

O

HOOC

O

H N

N H

HN

COOH

HOOC

O

H N

H N

N H

O COOH

O

N H

O

COOH

TPE-GFFYK(DVEDEE-Ac) O

H N

H N

OH

S O

O

H N O

HOOC

HOOC H N

N H

HN

N H

O

O

COOH

TPE-K(DVED-Ac)

B

OH

H N

H N C S

O N H

O

H N

H N

N H

O

COOH

O N H

O

S

O

H N

S

N H

O

COOH

Synthesis, Characterization and Design Principle of the Probes. It has been established that the peptide derivative of GFFY capped with an aromatic group has good self-assembly capability, which is able to self-assemble into nanostructures in an ordered manner.34-36 In this work, TPE has been demonstrated for the first time as an aromatic capping group of GFFY to yield TPE-GFFY with orderly self-assembling capacity, which was used to substitute TPE to fabricate AIE fluorescent light-up bioprobe. It is hypothesized that in aggregate state, the TPE molecules themselves would be randomly distributed in the aggregates, whereas upon methodical selfassembly of TPE-GFFY, more orderly arrangement of TPE could be achieved (Scheme 1A). If this is the case, it is reasonable to expect that the fluorescence intensity of TPE-GFFY aggregates would be higher than that of TPE aggregates at the same TPE concentration. This is because more orderly and regular array of TPE molecules is expected to lead to closer and tighter intermolecular steric interactions, which will more efficiently restrict the intramolecular rotations of phenyl rings in TPE. Using as a metaphor, it is just like putting many butterflies into a box. If the butterflies are arrayed with good or-

H N

O OH

O COOH

TPE-GFFYE-SS-EE COOH H N

H N S

O S

S

N H

TPE-SS-EE

H N

O

O

N H

COOH

COOH

TPE-GFFYK(DVEDEE-Ac) Probe for Sensitive and Selective Detection of Caspase-3. TPE-GFFYK(DVEDEE-Ac), TPE-K(DVED-Ac) and TPE-ITC exhibit similar absorption spectra in water (Figure S5 in the SI). Additionally, as shown in Figure 1A, the water-soluble TPE-GFFYK(DVEDEE-Ac) and TPE-K(DVED-Ac) (2 µM) do not emit in piperazineN,N’-bis(2-ethanesulfonic acid) (PIPES) buffer, but switch to fluoresce when incubated with caspase-3 (70 pM). The fluorescence enhancements of the two probes in the presence of caspase-3 should be ascribed from the enzyme cleavage at the carboxylic terminal of DEVD (Figure S6 in the SI). After removal of the hydrophilic moiety, the TPE-GFFYK and TPE-K residues form nanostructures, respectively (Figures 1B and 1C), activating the TPE fluorescence.



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Noteworthy, as shown in Figure 1A, after hydrolysis by caspase-3, the fluorescence intensity of TPE-GFFYK residues is ~6.9-fold and ~4.0-fold higher than that of TPE-K residues and TPE-ITC aggregates, respectively, at the same TPE concentration. The transmission electron microscopy (TEM) observations indicate that the TPE-GFFYK residues are able to form filamentous network structures with width of ~40 nm and length of micrometer levels (Figure 1B). In sharp comparison, both the TPE-K residues and TPE-ITC aggregates form nanoparticles with a mean diameter of 75 nm and 105 nm, respectively (Figures 1C and 1D). It has been reported that GFFY with an aromatic capping group often regularly self-assemble into nanofibrous network.34-38 The difference between TPEGFFYK and TPE-K residues in morphology reveals their different intrinsic molecular arrangements and verify the orderly self-assembly of TPE-GFFYK molecules. These results together also demonstrated that by virtue of such orderly selfassembly, the caspase-3-triggered fluorescence turn-on ratio is significantly enhanced for TPE-GFFYK(DVEDEE-Ac) probe.

Figure 1. (A) Photoluminescence (PL) spectra of TPE-ITC aggregates as well as TPE-GFFYK(DVEDEE-Ac) and TPEK(DVED-Ac) with and without treatment of caspase-3. [TPEGFFYK(DVEDEE-Ac)] = [TPE-K(DVED-Ac)] = [TPE-ITC] = 2 µM; [caspase-3] = 70 pM. Inset: the photographs taken under illumination of a UV lamp. TEM images of (B) TPEGFFYK(DVEDEE-Ac) and (C) TPE-K(DVED-Ac) after treatment with caspase-3 and (D) TPE-ITC aggregates. (E) Plot of I/I0 against caspase-3 concentration. I and I0 are the PL intensities in the presence and absence of the enzyme, respectively. (F) Plot of I/I0 versus different proteins. I and I0 are the PL intensities at the protein concentration of 70 and 0 pM, respectively. The data are presented as mean ± standard deviation (n = 3). ** P < 0.01, in comparison between the two probes indicated made by Student’s t-test. [probe] = 2 µM for (E) and (F).

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The fluorescence turn-on ratios (I/I0) of TPEGFFYK(DVEDEE-Ac) and TPE-K(DVED-Ac) (2 µM) after treatment with caspase-3 were plotted as a function of caspase-3 concentration, as displayed in Figure 1E. The I/I0 of TPEGFFYK(DVEDEE-Ac) increases linearly (R2 = 0.99851) with the increase in caspase-3 concentration ranging from 0 to 70 pM, indicating the potential utility of the probe for caspase-3 quantification. In contrast, the I/I0 of TPE-K(DVED-Ac) is much lower than that of TPE-GFFYK(DVEDEE-Ac) at each enzyme concentration. In addition, the fitting linear line for TPE-K(DVED-Ac) is not that perfect with an R2 = 0.88392 due to the relatively small fluorescence enhancement at the low probe concentration of 2 µM. The limit of detection (LOD) for TPE-GFFYK(DVEDEE-Ac) is calculated to be 0.54 pM based on the data in Figure 1E by utilizing a 3-sigma method,39 which is far lower than that of TPE-K(DVED-Ac) (3.50 pM). The determination of LOD values is discussed in detail in the SI. This result substantiates that as compared to TPE-K(DVED-Ac), TPE-GFFYK(DVEDEE-Ac) exhibits much larger fluorescent turn-on ratio and higher sensitivity in sensing caspase-3. To the best of our knowledge, 0.54 pM represents the lowest LOD value in caspase-3 detection compared with the currently reported fluorescent probes,40-42 indicating the ultrahigh sensitivity of TPE-GFFYK(DVEDEE-Ac). The specific recognition ability of the probes to caspase-3 was subsequently investigated by treating TPEGFFYK(DVEDEE-Ac) and TPE-K(DVED-Ac) with a variety of proteins, respectively, under identical concentrations. The results displayed in Figure 1F and Figure S7 in the SI reveal that the reference proteins including bovine serum albumin (BSA), lysozyme, trypsin, pepsin, alkaline phosphatase, proteinase K, cathepsin B, papsin, tax-interacting protein-1 can hardly switch on the fluorescence for both probes, indicating their excellent selectivity. Apoptosis Imaging in Cells with TPEGFFYK(DVEDEE-Ac). The utilization of both probes in apoptosis imaging in cancer cells was next studied using CLSM. After incubation of the HeLa cancer cells with 0.2 µM of TPE-GFFYK(DVEDEE-Ac) and TPE-K(DVED-Ac) at 37 o C for 2 h, respectively, the drug staurosporine (STS) was employed to induce the cell apoptosis, which was followed by imaging with CLSM. As shown in Figure 2, negligible fluorescence signal is detected in both the TPEGFFYK(DVEDEE-Ac)-treated and TPE-K(DVED-Ac)treated healthy HeLa cells. After cell apoptosis induced by STS, bright fluorescence signal can be observed within the TPE-GFFYK(DVEDEE-Ac)-treated apoptotic cells (Figure 2A), whereas very few discrete dots with low fluorescence are localized in the TPE-K(DVED-Ac)-treated apoptotic cells (Figure 2B). Moreover, the fluorescence turn-on of TPEGFFYK(DVEDEE-Ac) is significantly impeded when the cells are pretreated with an efficient caspase-3 inhibitor, 5[(S)-(+)-2-(methoxymethyl)pyrrolidino] sulfonylisatin (Figure S8 in the SI). This result indicates that TPEGFFYK(DVEDEE-Ac) can specifically respond to caspase-3 in cells and is much more sensitive than TPE-K(DVED-Ac) in imaging cell apoptosis. In addition, TPE-GFFYK(DVEDEEAc) and TPE-K(DVED-Ac) exhibit low cytotoxicities against both HeLa cancer cells and NIH/3T3 fibroblast cells (Figure S9 in the SI), indicating that they are safe probes for cellular imaging.


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Analytical Chemistry played in Figure 3A. TPE-GFFYE-SS-EE and TPE-SS-EE open the radiative pathways with great fluorescence enhancements. As expected, the fluorescence intensity of the residues of GSH-treated TPE-GFFYE-SS-EE is ~4.9 times higher than that of the residues of GSH-treated TPE-SS-EE, showing the larger fluorescent turn-on ratio and much higher sensitivity in detecting GSH. The cleavage of the disulfide bond in each probe after reaction with GSH was verified by MS analysis (Figure S15 in the SI). The TEM results displayed in Figures 3B and 3C reveal the distinctly different morphologies of the residues of TPE-GFFYE-SS-EE (network nanostructures) and TPE-SS-EE (nanoparticles) after incubation with GSH. Moreover, the three amino acids, glycine, glutamate, and cysteine contained in GSH were also added to the PBS solution of both TPE-GFFYE-SS-EE and TPE-SS-EE, respectively. As shown in Figure 3D, only cysteine with free thiol group can switch on the probe fluorescence, further demonstrating that the GSHtriggered fluorescence activation is due to the reaction between the free thiol in GSH and the disulfide bond in probe.

Figure 2. CLSM fluorescence and transmission images of healthy and apoptotic HeLa cells after treatment with 0.2 µM of (A) TPE-GFFYK(DVEDEE-Ac) and (B) TPE-K(DVEDAc), respectively. TPE-GFFYE-SS-EE Probe as Another Proof-of-Concept. To further confirm the higher sensitivity beneficial from the integration of the self-assembling peptide of GFFY, the probe of TPE-GFFYE-SS-EE containing a thiol-specific cleavable disulfide linker (-SS-) was designed as another proof-ofconcept with TPE-SS-EE as the control. Scheme 2B shows the chemical structures of TPE-GFFYE-SS-EE and TPE-SS-EE, which were prepared via the synthetic routes shown in Schemes S3 and S4 and characterized by 1H NMR, LC-MS and UV-vis, respectively (Figures S10-S14 in the SI). In addition, as shown in Figure 3A, both the TPE-GFFYE-SS-EE and TPE-SS-EE (5 µM) are in a completely fluorescence “off” state in phosphate buffered saline (PBS) solution as they exist as molecular species that lead to facile intramolecular rotations of phenyl rings of TPE. The glutathione (GSH) with free thiol group was then used as a reductant to cleave the disulfide bond in the probe.43 After incubation of GSH (250 µM) with each probe (5 µM) in PBS buffer for 3 h, the emission spectra were measured, as dis-

Figure 3. (A) PL spectra of TPE-GFFYE-SS-EE and TPE-SSEE in the absence and presence of GSH in PBS buffer. [TPEGFFYE-SS-EE] = [TPE-SS-EE] = 5 µM; [GSH] = 250 µM. TEM images of (B) TPE-GFFYE-SS-EE and (C) TPE-SS-EE after treatment with GSH. (D) Plot of I/I0 versus glutamate,



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glycine, cysteine and GSH. I and I0 are the PL intensities at the analyte concentration of 250 and 0 µM, respectively. [probe] = 5 µM. The data are presented as mean ± standard deviation (n = 3). ** P < 0.01, in comparison between the two probes indicated made by Student’s t-test. CLSM fluorescence and transmission images of A549 cancer cells treated with 1 µM of (E) TPE-GFFYE-SS-EE and (F) TPE-SS-EE, respectively. To test whether TPE-GFFYE-SS-EE possesses higher sensitivity than TPE-SS-EE in imaging intracellular thiol levels in cancer cells, A549 lung carcinoma cells were incubated with 1 µM of TPE-GFFYE-SS-EE and TPE-SS-EE, respectively. Figure 3E shows the CLSM images of TPE-GFFYE-SS-EEtreated A549 cells. Bright blue fluorescence is homogenously distributed in the cytoplasm, probably in both cytosol and endosomes, indicating the fluorescence light-up of the probe inside the cells. In contrast, at the same probe concentration, only weak fluorescent signal can be detected in the TPE-SSEE-treated A549 cytoplasm (Figure 3F). It is noted that under the same imaging conditions, no autofluorescence is observed from the A549 cell itself without any probe incubation (Figure S16 in the SI). These results substantiate that the AIE light-up probe with integrating the self-assembling peptide is much more sensitive in visualizing and monitoring intracellular thiols. Additionally, the cytotoxicity studies suggest that TPEGFFYE-SS-EE and TPE-SS-EE can be safely applied in cell experiments (Figure S17 in the SI).

CONCLUSION In conclusion, we report a simple and effective strategy to achieve highly sensitive AIE light-up probes via incorporating self-assembly peptide GFFY. The AIE fluorescent light-up probe of TPE-GFFYK(DVEDEE-Ac) was designed and synthesized. TPE-K(DVED-Ac) was also prepared as a control. The two probes are non-emissive in aqueous media; however, in the presence of caspase-3, the carboxylic terminal of DEVD is cleaved, which significantly switches on the fluorescence of both probes due to the removal of hydrophilic moieties. After enzyme-catalyzed hydrolysis, the TPE-GFFYK residues regularly self-assemble into filamentous network nanostructures, whereas TPE-K residues form nanoparticles. In contrast to TPE-K residues, the more orderly self-assembly of TPEGFFYK residues results in more effective restriction of intramolecular rotations of phenyl rings in TPE. This endows TPEGFFYK(DVEDEE-Ac) with larger fluorescent turn-on ratio, lower enzyme detecting limitation and thus higher sensitivity in detecting and imaging caspase-3 in both solutions and cancer cells, as compared to previously reported TPE-K(DVEDAc). A detection limit as low as 0.54 pM can be achieved for TPE-GFFYK(DVEDEE-Ac) towards caspase-3, which represents the lowest LOD value in caspase-3 detection compared with the currently reported fluorescent probes, to the best of our knowledge. Furthermore, the probe of TPE-GFFYE-SSEE was also utilized as another proof-of-concept, which also shows considerably enhanced sensitivity in detecting GSH and visualizing intracellular thiol levels compared with the control probe TPE-SS-EE. This study thus demonstrates a generic method to achieve highly sensitive AIE fluorescent light-up probes through simply employing GFFY to induce ordered self-assembly of AIEgens.

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Supporting Information Synthesis and characterization of the probes, UV-vis spectra of the probes, confocal images of drug-induced HeLa cells pretreated with caspase-3 inhibitor before TPE-GFFYK(DVEDEE-Ac) staining, cytotoxicity study of the probes, experimental section and determination of LOD values. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (D. Ding) * E-mail: [email protected] (Z. Yang) * E-mail: [email protected] (B. Liu)

Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (2015CB856503), the NSFC (31571011, 81301311, 81220108015 and 51222303), the PCSIRT (IRT13023), the Science & Technology Project of Tianjin of China (No. 15JCYBJC29800), the Singapore National Research Foundation (R-279-000-444-281), the Singapore-MIT Alliance for Research and Technology (SMART) Innovation Grant (R279-000-378-592), and the Research Grants Council of Hong Kong (HKUST/CRF/10 and N_HKUST620/11).

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Peptide-Induced AIEgen Self-Assembly: A New Strategy to Realize

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