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Bio-inspired peptide for imaging Hg distribution in living cells and zebrafish based on coordination-mediated supramolecular assembling Shilang Gui, Yanyan Huang, Fang Hu, Yulong Jin, Guanxin Zhang, Deqing Zhang, and Rui Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00059 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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Analytical Chemistry

Bio-inspired peptide for imaging Hg2+ distribution in living cells and zebrafish based on coordination-mediated supramolecular assembling Shilang Gui,†,‡ Yanyan Huang,*,†,‡ Fang Hu,†,‡ Yulong Jin,†,‡ Guanxin Zhang,†,‡ Deqing Zhang, †,‡

and Rui Zhao*,†,‡

† Beijing National Research Center for Molecular Sciences, CAS Key Laboratories of Analytical Chemistry for Living Biosystems and Organic Solids, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ‡ University of Chinese Academy of Sciences, Beijing, 100049, China

* Email: [email protected], [email protected]

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ABSTRACT Peptides with modular structure provide a tailorable platform for constructing responsive supramolecular assemblies, which are attractive as functional biomaterials and smart sensors. In this work, the feasibility of regulating small peptides assembly with molecular design and metal ion recognition was demonstrated. Tripeptides were designed and found to have diverse response and self-assembly behavior to Hg2+. The incorporation of an aggregation-induced emission fluorophore TPE enabled the visualization of Hg2+ recognition and the assembly phenomenon. A structural analogue (Pep2) to γ-glutathione was identified with high specificity and nanomolar response to Hg2+ both in buffer solution and living cells. Driven by the coordination force and non-covalent intra-molecular stacking, assembling of twisted nanofibers from Pep2-TPE and Hg2+ were observed. Benefiting from its biocompatibility, fast and switchable fluorescence response, Pep2-TPE was applied for imaging and monitoring Hg2+ distribution in living cells and zebrafish. With good permeability to plasma membrane and tissues, Pep2-TPE indicated the preferential distribution of Hg2+ in cell nucleoli and brain of zebrafish, which is related with the deleterious effect of inorganic mercury in living biosystems.


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INTRODUCTION Supramolecular self-assembly has attracted considerable research interests as a bottom-up approach for the fabrication of nanomaterials with ordered structures and multi-functionalities.1-4 Peptides with amphiphilic property, built from optional amino acids, capable of accommodating various non-covalent interactions provide a highly programmable library for the fabrication of supramolecular architectures.5-8 Nowadays, peptide-based self-assemblies with different morphologies have been reported ranging from nanoparticles to amyloid fibers.9,10 Remarkable applications have been achieved in the fields of biomedicine, electronic engineering, catalysis and biosensors.11-14 Metal ion coordination has been proven to be a powerful driving force to induce molecular assembly, which motivates the production of polymer and crystalline materials such as metal organic frameworks (MOFs).15-17 As building units, peptides not only can provide multiple metal binding sites,13,18,19 but also bring distinct properties as chirality and specific biorecognition.20-22 Owing to their biocompatibility, structural simplicity, as well as the ease in chemical synthesis and modification, recent research progress is made towards the discovery of small peptides.23-25 Moreover, integrating activatable signal and specific stimuli with metalpeptide assembling behavior becomes attractive, which is particularly essential for biosensing and biomedical applications.20,26,27 Mercury featuring in high bioaccumulation, biomagnification and persistence in environment is a great threat to human health.28,29 Capable of easily passing biological membranes, mercury can cause birth defect and damages to various organs including lung, kidneys, central nervous system, and endocrine system.30-32 Although the toxicity of this metal is recognized, the biodistribution and the direct relations with many diseases especially in its inorganic forms still remain unclear and need further investigations.33 Given that mercury is a soft Lewis acid, a number of ligands capable of strongly coordinating Hg2+ have been employed as affinity ligands for Hg2+ detection and quantitation, such as nucleosides, peptides and sulfur-containing compounds.34-37 However, there are still some challenging tasks including interferences from other heavy metal ions thus limited selectivity, delay in response, and poor compatibility with insitu monitoring. Therefore, developing flexible systems is urgently demanded for real-time, specific, and responsive monitoring Hg2+, especially for in vivo tracing and evaluating biological functions of this ion.


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Nature provides plentiful resource for molecular recognition. Glutathione (γ-Glu-Cys-Gly, γGSH), an important tripeptide with an unusual gamma peptide linkage, acts as a cellular scavenger of various heavy metal ions.38,39 γ-GSH has also been found to form assemblies in the presence of inducers.40-42 In this study, inspired by the structure of γ-GSH, tripeptides were designed for the construction of Hg2+-responsive supermolecular assemblies. The incorporation of an aggregation-induced emission (AIE) fluorophore43-46 to the peptides served as the switchable reporter for Hg2+ and indicator of the assembly phenomenon. By using the sequence flexibility of peptides, the selectivity of assembly behavior was expected to modulate from nonspecific towards highly specific to Hg2+. The changes in peptide structure would affect the coordinating affinity, fluorescence response, supramolecular assembly and thus the morphology of nano-architectures. Taking advantage of the good biocompatibility of peptides, the in vivo application of the Hg2+-mediated peptide assembly and fluorescence turn-on effect was carried out, which could provide information on the real-time distribution of this ion in living cells and zebrafish.

EXPERIMENTAL SECTION Synthesis. Tetraphenylethylene (TPE) conjugated peptides were synthesized using solid phase synthesis approach (Scheme S-1). FMOC-Gly-Wang resin (Gly loading: 0.698mmol/g) was used as the starting material. Piperidine (20% in DMF) was used as the de-protection reagent. 4methylmorpholine was used as the activating reagent, and HBTU was used as the coupling agent. After peptide elongation, TPE-COOH was added to the amino terminals of the peptides under the same conditions. Finally, the TPE-conjugated peptides were cleaved from the resins with a freshly prepared cocktail (94% TFA, 2.5% H2O, 2.5% EDT and 1% TIPS as scavengers). The crude products were purified on a Shimadzu HPLC system (Kyoto, Japan) using a C18 column (Dikma, 10×250 mm i.d.). Mobile phase: water with 0.1% TFA and CH3CN with 0.1% TFA. After purification, the compounds were analyzed with an Ultimate 3000 UHPLC system (ThermoFisher, San Jose, CA, USA) and characterized with high-resolution mass spectrometer (HR MS) equipped with a MALDI source (Solarix 9.4T AS FTICR MS, Bruker Daltonics, Bremen, Germany).


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Fluorescence measurement. The stock solutions of different metal ions (0.25 mM) were prepared with ultra-pure water respectively. Stock solutions of TPE-conjugated peptides were prepared with dimethyl sulfoxide to a concentration of 1 mM. For a typical detection, 10 µL of peptide stock solution was mixed with 10 µL of the Hg2+ stock solution, followed by the dilution with appropriate amounts of phosphate buffered saline (PBS) to a final volume of 500 µL. The fluorescence spectrum was recorded on a Hitachi F-4600 fluorometer (Tokyo, Japan). The excitation wavelength was 330 nm, and the emission was collected from 350 to 625 nm. Electron microscopic characterization. The morphology of Hg2+-mediated peptide assemblies was characterized with TEM and SEM. For a typical sample preparation, the mixed solution of Hg2+ (5µM) and Pep2-TPE (20µM) was dipped on cover slip. After being left under atmosphere for dryness, the samples were subjected for TEM observation. For SEM characterization, sample solutions were dipped on cover slip, dried under vacuum and coated by a thin layer of platinum before observation. Cell imaging. HeLa cells were seeded into glass bottom culture dishes at a density of 1.0×105 cells/mL and cultured overnight for adhesion. The adherent cells were washed with buffer three times and loaded with Hg2+ at 37 oC for 10 min. After removal of free Hg2+ by washing the cells with buffer solution, Pep2-TPE was added to incubate with the cells for 20 min. Fluorescence imaging was performed on an Olympus IX83 fluorescence microscope. A high pressure mercury burner (USH-1030L, 100W, Olympus, Japan) was used as the excitation source. The objective used for imaging was a UPLSAPO 100× oil-immersion objective NA 1.40 (oil) (Olympus). Excitation filter: BP325-375 (Olympus), emission filter: BA420 (Olympus). Image processing and analysis were performed on Olympus software (cellsens standard) and Image-Pro Plus 5.0. For comparison, all of the parameters of the microscope were set to be the same for the corresponding samples. All data were from three separated measurements. Fluorescence imaging of Hg2+ in zebrafish. Zebrafish were obtained from the National Zebrafish Resources of China. Zebrafish were grown in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 5-10% methylene blue; pH 7.5). For fluorescence imaging, the embryos or the 5-dayold zebrafish were incubated with Hg2+ in E3 embryo media for 10 min. After washing with Lock’s buffer (pH 7.4) to remove the remaining Hg2+, the embryos or the larvae were further


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treated with Pep2-TPE for 30 min. The fluorescence images were collected directly an Olympus IX83 fluorescence microscopy without further washing steps. A high pressure mercury burner (USH-1030L, 100W, Olympus, Japan) was used as the excitation source. The objective used for imaging was a PLANAPO N 2× objective NA 0.08 (Olympus). Excitation filter: BP325-375 (Olympus), emission filter: BA420 (Olympus). Image processing and analysis were performed on Olympus software (cellsens standard). For comparison, all of the parameters of the microscope were set to be the same for the corresponding samples. All animal operations were in accord with the institutional ethics committee regulations and guidelines on animal welfare.

RESULTS AND DISCUSSION Design of peptides. As the starting structure, γ-GSH (Pep1 in Scheme 1) has an unusual gamma peptide linkage between the carboxyl group of glutamate side chain and the amine group of cysteine. In nature, peptide bond however normally forms by the alpha carboxyl group and the alpha amino group between two residues. With these consideration, Pep2 (Glu-Cys-Gly) was designed with exactly the same amino acid sequence with γ-GSH but the alpha linkage between glutamic acid (Glu) and cysteine (Cys) (Scheme 1). Since thiol and carboxylic acid are frequently found in the metal binding sites of many proteins,35 the thiol and carboxylic acid side chains in Pep3-Pep5 were respectively substituted to investigate their functions for Hg2+ recognition (Scheme 1). As a result, Pep3-Pep5 were designed with the sequences of Ala-CysGly, Glu-Ala-Gly and Ala-Ala-Gly, respectively. To provide detectable signal for Hg2+ recognition and peptide assembly, tetraphenylethylene (TPE), a typical AIE fluorophore was chosen. The AIE effect of TPE is expected to introduce the OFF-ON responsive fluorescence, thus allows highly sensitive detection and sensing. The TPEmodified peptides (Pep1-TPE ~ Pep5-TPE) were synthesized using the solid phase synthesis (Scheme S-1) and were characterized with HPLC and HR MS (Figure S-1 and S-2, Supporting Information).


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Scheme 1. Structures of designed peptides Pep1-Pep5 and their conjugates with TPE. Hg2+-induced fluorescence turn-on. Aim at applying the peptide probe for Hg2+ analysis in biological systems, phosphate buffered saline (PBS) was used as the solvent to mimic the physiological environment. The additive DMSO (2%) is also biocompatible. Benefiting from high hydrophilicity of the peptides, Pep1-TPE ~ Pep5-TPE kept fluorescence silent in PBS (2% DMSO) (Figure 1), which provides the prerequisite for the fluorescence turn-on response to Hg2+. Upon the addition of Hg2+, strong blue fluorescence was emitted from Pep2-TPE solution (Figure 1). The intensity at 470 nm was 130-fold enhanced. Such OFF-ON switch of the fluorescence can be easily detected with naked eye under UV light (Figure 1b). Pep1-TPE also responded to the addition of Hg2+, which is consistent with the chelating ability of γ-GSH to heavy metal ions. However, the fluorescence enhancement was much weaker (Figure 1a and b). These results demonstrate that simple change from gamma peptide linkage to alpha linkage significantly enhances the binding affinity of Pep2-TPE with Hg2+. For Pep3-TPE ~ Pep5-TPE, almost no fluorescence was detected after the addition of Hg2+, suggesting their low binding affinity (Figure 1a and b). Although they all have the alpha peptide linkages as Pep2-TPE, the mutation in the amino acid compositions diminishes the chelating with the ion. Considering their structures, both thiol and carboxyl side chains are essential for the recognition of Hg2+.


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Figure 1. (a) Fluorescence spectra of Pep1-TPE~Pep5-TPE (20 µM) responding to Hg2+ (5 µM), solvent: PBS (2% DMSO). (b) Photographs of Pep1-TPE~Pep5-TPE solutions with and without the presence of Hg2+. I: Pep1-TPE, II: Pep2-TPE, III: Pep3-TPE, IV: Pep4-TPE, V: Pep5-TPE. [peptide-TPE] = 20 µM, [Hg2+] = 5 µM, Solvent: PBS (2% DMSO). (c) Fluorescence variations of Pep2-TPE responding to different metal ions, respectively. Insert: fluorescence response of Pep1-TPE and Pep2-TPE to the mixtures of metal ions. mixed ions = Pb2+, Al3+, Cu2+, Ni2+, Li+, Mg2+, Co2+, Ca2+, Cr3+, K+, Na+, Fe3+, Ba2+, Cd2+, Zn2+, Mn2+, Fe2+ and Ag+. [Peptide-TPE] = 20 µM, each [metal ion] = 5 µM, solvent: PBS (2% DMSO). (d) Fluorescence spectra of Pep2-TPE (20 µM) in the presence of Hg2+ with varied concentrations. Insert: plot of the fluorescence intensity of Pep2-TPE (20 µM) versus Hg2+ concentration, solvent: PBS containing 2% DMSO. To examine the selectivity of the peptides, 18 metal ions with different valences were mixed with Pep2-TPE instead of Hg2+. None of these metal ions can light up the fluorescence of Pep2TPE under this condition (Figure 1c). Hg2+ can be easily discriminated by the distinct blue fluorescence from Pep2-TPE either existing individually or coexisting with other metal ions in complicated samples (Figure 1c). In comparison, Pep1-TPE showed poor selectivity in identifying Hg2+. Pep1-TPE responded to metal ion mixtures with almost the same fluorescence intensity no matter Hg2+ is present or not (Figure 1c). Hence, simple modification of peptide bond to alpha linkage brings high specificity to Pep2-TPE for Hg2+ recognition. Kinetic study showed that fluorescence from the binding of Pep2-TPE and Hg2+ reaches the maximum in 1 min (Figure S-3), manifesting the rapid binding and signal release. The effect from pH was also examined by pH titration assay (Figure S-4). In the pH range of 6.5–7.5, the


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strongest fluorescence response from Pep2-TPE to Hg2+ was detected. The fast binding kinetics and neutral working pH of Pep2-TPE towards Hg2+ is appealing for real-time monitoring in living biosystems. The dependence of the fluorescence of Pep2-TPE on Hg2+ concentration (Figure 1d) shows that saturation of the binding can be obtained with 0.5 equiv. of Hg2+. A detection limit of 1.9 nM (signal/noise = 3) towards Hg2+ can be estimated accordingly (Figure S-5), which was much lower than many previously reported fluorescence probes.35,36,47-49 Hg2+-mediated self-assembly. The fluorescence emission of AIE fluorophores usually denotes the formation of nano-assemblies. The mixed solution of Pep2-TPE and Hg2+ was firstly characterized with dynamic light scattering (DLS). The appearance of two peaks centered at 51 nm and 4800 nm indicates two dramatically varied dimensions (Figure 2a). This is most probably ascribed to the formation of nanofibers from Pep2-TPE in the presence of Hg2+ as observed with TEM (Figure 2b). In TEM images, the average diameter of nanofibers is 50 nm, which is in accordance with the size from the first peak in DLS (51 nm). Considering the fibrous morphology, the peak centered at 4800 nm most probably represents the length of the nanofibers, which may stretch for several micrometers. Under fluorescence microscopy, emission of bright blue fluorescence was detected for the nanofibers (Figure 2f), confirming the self-assembly from Pep2-TPE unit. In the control experiments without Hg2+, no nanofiber was generated in both TEM and fluorescence microscopic characterization (Figure S-6). These results demonstrate the important role of Hg2+ in mediating the supramolecular assembly of Pep2-TPE.


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Figure 2. (a) DLS analysis of Pep2-TPE (20 µM) in the presence of Hg2+ (5 µM). (b-d) TEM characterization of Hg2+-induced nanofibers assembled from Pep2-TPE. Scale bar: 200 nm. Yellow arrows indicate the twisted structure. (c, d) Zoom-in TEM images of zone 1 and 2 in Figure 2b. (e) TEM characterization of Hg2+-induced nanofibers assembled from Pep1-TPE. Scale bar: 200 nm. (f, g) Fluorescence microscopic imaging of Hg2+-induced nanofibers assembled from Pep2-TPE (f) and Pep1-TPE (g). Scale bar: 10 µm. (h) SEM characterization of Hg2+-induced nanofibers assembled from Pep2-TPE. Scale bar: 200 nm. Yellow arrows indicate the formation of larger twisted fibers. In the presence of Hg2+, Pep1-TPE also formed nanofibers as detected with TEM and fluorescence microscopy (Figure 2e and g). However, the diameter of the fibers (22 nm) is much smaller than those assembled from Pep2-TPE. Accordingly, the fluorescence intensity from Hg2+/Pep1-TPE fibers was obviously weaker under the same conditions. This is in consistent with the result from fluorescence spectra (Figure 1a) and may be attributed to the lower aggregation degree of TPE moiety. The difference in peptide bond not only significantly affect the coordination ability towards Hg2+, but also the self-assembly behaviors. For Pep3TPE~Pep5-TPE, none of them can assemble to nanofibers (Figure S-7). The substitution even in one residue disables their coordination with Hg2+ and supramolecular assembly. Investigation of Hg2+-induced assembly mechanism. With close observation to Pep2-TPEassembled nanofibers, left-handed chiral twist can be found (Figure 2b-d). Moreover, further twist of smaller nanofibers to form lager left-handed helical structure was detected in SEM image (Figure 2h). Such sequential assembly of small fibers to larger and brighter ones was also imaged with fluorescence microscopy (Figure 2f). Circular dichroism (CD) spectroscopy, a powerful tool for conformation and chiral characterization, was employed to examine the supramolecular assembly (Figure 3a). In the presence of Hg2+, the CD spectrum displays a positive band at 310 nm and a negative band at 357 nm with a crossover at 330 nm which is identical with the adsorption band of Pep2-TPE (Figure S-8). This phenomenon indicates the existence of chirality in the assembled structure.41,50 The two sharp peaks are signature of Cotton effects and caused by the coupling low-energy ligand-to-metal charge transfer bands in the extended coordination systems,41 such as fibular structure in this study. Left-handed chiral or helix is ubiquitously adopted by biomolecules in nature such as amino acids, peptide and DNA.


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For Pep2-TPE, the two L-amino acids Glu and Cys in the peptide sequence bring two chiral centers. These results confirm the interactions between Pep2-TPE and Hg2+, and left-handed molecular chirality of amino acids was delivered to the twisted configuration of assembled nanofibers.

Figure 3. (a) CD spectra of Pep2-TPE (20 µM) with and without the existence of Hg2+ (5 µM). (b) HR MS characterization of the coordination complex from Pep2-TPE and Hg2+ (ESI, negative mode). The calculated m/z for [2L+Hg-4H]2- is 794.1998; the calculated m/z for [2L+Hg-3H]- is 1589.4074. (c) Possible process for the supermolecular assembly of fluorescent nanofibers from the coordination complex of Pep2-TPE and Hg2+. The coordination complex was further characterized with high resolution MS (HR MS), fluorescence titration and microscopic FT-IR. In HR MS spectrum (Figure 3b), the m/z signals (m/z 794.2010 and 1589.4091) corresponding to the complexes of [2L+Hg2+] (L = Pep2-TPE) suggest that the binding stoichiometry of Pep2-TPE with Hg2+ is 2:1. By continuous variation of Hg2+ mole fraction while keeping the total concentration of Hg2+ and Pep2-TPE at 30 µM, a peak was reached at a molar fraction of 0.33 in the Job’s plot, confirming the formation of 2:1 complex between Pep2-TPE with Hg2+ (Figure S-9). Based on this binding stoichiometry, the association constant (Ka) was determined as 5.60×1011 M-2 (Figure S-10), which reveals the strong binding between Pep2-TPE and Hg2+. For FT-IR characterization, the disappearance of the S-H bond vibration, shifts of C=O and C-O stretching vibrations of Pep2-TPE exhibited the involvement of thiol and carboxyl groups in Pep2-TPE for Hg2+ chelating (Figure S-11). Based on the above analysis, the Hg2+-induced assembly is speculated in Figure 3c. In the presence of Hg2+, the side chains thiol and carboxyl groups in Pep2-TPE participated in the coordination


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interactions, leading to the formation of the building unit [2L+Hg2+]. Driven by non-covalent forces such as electrostatic interaction, hydrogen bonds between amide and carboxyl groups, and π-π stacking between the benzene rings in TPE moiety, the building unit further self-assembled into fibular structure. Due to the chiral nature of Pep2 molecule, the nanofibers distorted to one direction and formed the same left-handed twisted morphology. With adaptable surface morphology and residue groups available for non-covalent interactions, some nanofibers twisted with each other for the generation of larger fibers. Tracing Hg2+ in living cells. The specific fluorescence turn-on response to Hg2+ makes Pep2TPE potential for imaging and monitoring Hg2+ in living biosystems. Given that endogenous biothiol species including cysteine, homo-cysteine and γ-GSH cannot obviously interfere with the binding of Pep2-TPE with Hg2+ (Figure S-12), Pep2-TPE was firstly used to tracing Hg2+ in living cells. Hg2+-preloaded HeLa cells (cervical cancer cell line) were treated with Pep2-TPE solution. As shown in Figure 4a-d, bright blue fluorescence was emitted from Hg2+-loaded cells after 20-min incubation, suggesting fast and good membrane permeability of Pep2-TPE and its complexation with Hg2+ inside cells. In comparison, no fluorescence signal can be detected from HeLa cells only treated with Pep2-TPE (Figure 4a). It is also noticeable that even without any washing steps, the images were acquired with high signal-to-noise ratio. This can be ascribed to the AIE property of TPE moiety and the switchable fluorescence of Pep2-TPE responding specifically to Hg2+. To further confirm the binding of Pep2-TPE to Hg2+ in cells, competitive binding assays employing a classic Hg2+ chelator dimercaptosuccinic acid (DMSA) as the competitor were carried out. No fluorescence can be observed inside Hg2+-loaded cells after the treatment with DMSA neither before nor after the addition of Pep2-TPE (Figure S-13, Supporting Information). This phenomenon can be attributed to the occupation of Hg2+ by DMSA which blocked the binding from Pep2-TPE. The results from competitive binding assays verify the critical role of the binding between Pep2-TPE and Hg2+ for fluorescence emission, which provides the foundation for tracing Hg2+ biodistribution in living cells with the probe.


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Figure 4. (a-d) Fluorescence microscopic imaging of Hg2+ in living cells with Pep2-TPE (10 µM). (a) HeLa cells treated with Pep2-TPE only. (b-d) Hg2+-loaded HeLa cells imaged with Pep2-TPE. [Hg2+] = 2.5 µM (b), 5 µM (c) and 10 µM (d), respectively. Scale bar = 10 µm. (e) Effect of concentration on the subcellular distribution of Hg2+ based on pixel intensity analysis. (f-g) Sensitive detection of Hg2+ in HeLa cells with Pep2-TPE. Cells were pre-treated with Hg2+ at concentration of 1 µM (f) and 200 nM (g) respectively. The biodistribution of Hg2+ in living cells responding to varied exposure dosages were investigated with Pep2-TPE. At the Hg2+ dosage of 2.5 µM, the whole cell was emissive, suggesting the universal distribution of Hg2+ in cell (Figure 4b). Notably, brighter fluorescence was observed from nucleus, demonstrating stronger retention of Hg2+ in cell nucleus. Furthermore, the brightest emission was detected from nucleoli (Figure 4b and e), the largest structure in the nucleus. This is in accordance with the gene damage effect of Hg2+ towards cells.51,52 When raising Hg2+ concentration to 5 µM, the fluorescence from both cytoplasm and nucleus significantly intensified (Figure 4c), indicating increased cellular uptake of Hg2+. The distinct preference of Hg2+ for nucleus, especially nucleoli was still detected by significantly stronger fluorescence in these structures (Figure 4e). Further increase of Hg2+ dosage to 10 µM resulted in further intensified emission from HeLa cells. Although brighter emission was still obvious in nucleoli, the signal distribution in the whole cell tended to be even and similar in cytoplasm and nucleus (Figure 4e). To explore the sensitivity of Pep2-TPE for Hg2+ tracking in living cells, lower Hg2+ concentrations were examined (Figure 4f and 4g). At a concentration as low as 200 nM, bright fluorescence still can be observed inside cells with higher intensity in the nucleus. In comparison, higher Hg2+ concentrations were usually used for the treatment of the cells to enable the imaging


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detection of intracellular mercury ion in many previous work.33,35,53,54 By using Pep2-TPE as the indicator, these results provide direct proof for the multiple distribution of Hg2+ in different cell compartments, which is responsible for its complicated damage effects to cells. The favorable accumulation of Hg2+ in nucleus, particularly nucleoli structure can be informative for studying the cytotoxicity mechanism of Hg2+. The exposure to Hg2+ even in lower concentrations could firstly resulted in genetic deficiencies. Because of the critical function of nucleoli in ribosomes creation, the strong accumulation of Hg2+ in nucleoli denotes the possible roles of Hg2+ in interfering ribosome biogenesis and functions. Compared with the well-known toxicity of organic mercury, the adverse effect of inorganic mercury especially during low dosage exposure is unclear. Capable of responding to lower concentration of Hg2+ in cells, Pep2-TPE can be favourable for the toxicology of Hg2+. Peptides containing Cys residue may have the tendency to be oxidized. For Pep2-TPE, it can keep the original status without oxidation in working solution for at least 48 hours (Figure S-14). The disability of its oxidized product for Hg2+ discrimination further excluded the interference from oxidation during applications (Figure S-15~S-17). The low cytotoxicity of Pep2-TPE was demonstrated by cell viability assay (Figure S-18). The high Hg2+-affinity, stability, switchable fluorescence, good penetrability and biocompatibility of Pep2-TPE are appealing for long-term bioanalysis and in vivo applications. Imaging Hg2+ distribution in zebrafish. Because mercury pollution is usually aquatic, fishes are among the first victims from mercury exposure especially from inorganic mercury. To apply Pep2-TPE for monitoring Hg2+ in living biosystems, zebrafish, a well-established vertebrate model organism, was used for in vivo imaging. The distribution and deleterious effect of Hg2+ in zebrafish embryo (segmentation stagy) was firstly investigated (Figure 5a-c). For embryos with pre-exposure to Hg2+, blue fluorescence was emitted from chorion after Pep2-TPE treatment, while the yolk and cells almost remain dark. This result implies that Hg2+ were mainly retained by chorion and prevented from accessing and damaging the yolk and embryo cells. Such phenomenon is in consistent with the protective role of chorion against the toxic species. A further effort was made to apply Pep2-TPE for imaging Hg2+ in living zebrafish larvae. As shown in Figure 5d-f, 5-day-old zebrafish display very weak auto-fluorescence from the yolk sac. Similar image was also obtained for zebrafish only incubated with Pep2-TPE. Upon the presence


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of Hg2+, different distribution of fluorescence signal was detected by Pep2-TPE (Figure 5f). Bright blue fluorescence was observed in the brain and at the periphery of the eye, suggesting the strong accumulation of Hg2+ in these compartments and the abilities of Pep2-TPE to penetrate tissues and even the blood brain barrier. From the high signal-to-background image, Hg2+ may be most probably localized to the ventricular region and visual nervous system. Such result is in accordance with the reports on the damage effect of mercury to the central nervous system.31,33 These in vivo studies manifest that Pep2-TPE is promising for monitoring Hg2+ distribution in living biosystems and are informative for further investigations on the deleterious effect of Hg2+ to the complicated functions of brain.

Figure 5. Imaging analysis of the distribution of Hg2+ (20 µM) in zebrafish embryo (a-c) and zebrafish larvae (d-f) with Pep2-TPE (50 µM). (a, d) without any treatment, (b, e) treated with Pep2-TPE only, (c, f) Hg2+ pre-exposed embryo/larvae treated Pep2-TPE. Scale bar: 500 µm. CONCLUSION In summary, this work demonstrates the feasibility of regulating small peptides-based assembly with molecular design and biorecognition event. Tripeptides with subtle structural variety were designed and found to have different responses and self-assembly behaviors to Hg2+. Simple change in peptide bond or single residue resulted in a highly specific peptide Pep2-TPE towards Hg2+ recognition. Tuned by such structural differential, Hg2+-mediated supramolecular assemble of twisted nanofibers was observed for Pep2-TPE. Benefiting from its biocompatibility, high selectivity, fast and switchable response to Hg2+, Pep2-TPE was successfully applied for imaging and monitoring Hg2+ in living cells and zebrafish. Given the high toxicity of organic mercury and the versatile structure of peptides, our future effort will be devoted to the design of specific


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peptide probes for monitoring organic mercury in vivo. By using these probes for both organic and inorganic mercury species, the biodistribution of total mercury and their respective mechanism for toxicity could be investigated. The tailorable metal-peptide biocoordination systems responding to specific stimuli with activatable signal show great potential in bioanalytical and biomedical applications in living biosystems.

ASSOCIATED CONTENT

Supporting Information Materials and apparatus. HPLC and HR MS characterization, binding kinetics, HR MS identification of coordination complex, absorption spectrum of Pep2-TPE, fluorescence titration, FT-IR characterization of the coordination complex, effect of biothiols on the fluorescence response of Pep2-TPE to Hg2+, competitive binding assays in cells, and cytotoxicity evaluation of Pep2-TPE. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Corresponding Author *Phone: 86-10-62557910; Fax: 86-10-62559373 Email: [email protected], [email protected] ORCID Rui Zhao: 0000-0001-7191-9354 Yanyan Huang: 0000-0002-2930-6471 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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This work is supported by grants from National Natural Science Foundation of China (21475140, 21675161 and 21621062), Ministry of Science and Technology of China (2015CB856303) and Chinese Academy of Sciences. We thank Prof. Zhang Bo from National Zebrafish Resources of China for the donation of zebrafish. We also express our gratitude to Prof. Zhenwen Zhao from Institute of Chemistry, Chinese Academy of Sciences for his valuable help in MS analysis. REFERENCES (1) Tu, Y.; Peng, F.; Adawy, A.; Men, Y.; Abdelmohsen, L. K. E. A.; Wilson, D. A. Chem. Rev., 2016, 116, 2023-2078. (2) Bai, Y.; Luo, Q.; Liu, J. Chem. Soc. Rev., 2016, 45, 2756-2767. (3) Kim, Y.; Li, H.; He, Y.; Chen, X.; Ma, X.; Lee, M. Nat. Nanotechnol., 2017, 12, 551-556. (4) Huang, Y.; Liu, Z.; Liu, C.; Ju, E.; Zhang, Y.; Ren, J.; Qu, X. Angew. Chem. Int. Ed., 2016, 55, 6646-6650. (5) Mosquera, J.; Szyszko, B.; Ho, S. K. Y.; Nitschke, J. R. Nat. Commun., 2017, 8, 14882. (6) Wang, H.; Feng, Z.; Lu, A.; Jiang, Y.; Wu, H.; Xu, B. Angew. Chem. Int. Ed., 2017, 56, 75797583. (7) Hosseinkhani, H.; Hong, P.-D.; Yu, D.-S. Chem. Rev., 2013, 113, 4837-4861. (8) Li, J.; Du, X.; Hashim, S.; Shy, A.; Xu, B. J. Am. Chem. Soc., 2017, 139, 71-74. (9) Klinker, K.; Schaefer, O.; Huesmann, D.; Bauer, T.; Capeloa, L.; Braun, L.; Stergiou, N.; Schinnerer, M.; Dirisala, A.; Miyata, K.; Osada, K.; Cabral, H.; Kataoka, K.; Barz, M. Angew. Chem. Int. Ed., 2017, 56, 9608-9613. (10) Wang, Y.; Chen, C. H.; Hu, D.; Ulmschneider, M. B.; Ulmschneider, J. P. Nat. Commun., 2016, 7, 13535. (11) Reithofer, M. R.; Chan, K.-H.; Lakshmanan, A.; Lam, D. H.; Mishra, A.; Gopalan, B.; Joshi, M.; Wang S.; Hauser, C. A. E. Chem. Sci., 2014, 5, 625-630. (12) Zou, Q.; Abbas, M.; Zhao, L.; Li, S.; Shen, G.; Yan, X. J. Am. Chem. Soc., 2017, 139, 19211927. (13) Kim, S.; Kim, J. H.; Lee, J. S.; Park, C. B. Small, 2015, 11, 3623-3640. (14) Omosun, T. O.; Hsieh, M. C.; Childers, W. S.; Das, D.; Mehta, A. K.; Anthony, N. R.; Pan, T.; Grover, M. A.; Berland K. M.; Lynn, D. G. Nat. Chem., 2017, 9, 805-809. (15) Furukawa, H.; Cordova, K. E.; O'Keeffe, M.; Yaghi, O. M. Science, 2013, 341, 1230444. (16) Kondo, M.; Yoshitomi, T.; Seki, K.; Matsuzaka H.; Kitagawa S. Angew. Chem. Int. Ed., 1997, 36, 1725-1727. (17) Lian, X. Z.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J. L.; Banerjee, S.; Lollar, C.; Wang X.; Zhou, H. C.; Chem. Soc. Rev., 2017, 46, 3386-3401. (18) Zou, R.; Wang, Q.; Wu, J.; Wu, J.; Schmuck, C.; Tian, H. Chem. Soc. Rev., 2015, 44, 52005219. (19) Yan, X.; Zhu, P.; Li, J. Chem. Soc. Rev., 2010, 39, 1877-1890.


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Bio-inspired peptide for imaging Hg distribution in ... - ACS Publications

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