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Multifunctional gold nanoclusters-based nanosurface energy transfer probe for real-time monitoring of cell apoptosis and self-evaluating of pro-apoptotic theranostics Yong Li, Pei Li, Rong Zhu, Chao Luo, Hao Li, Shanfang Hu, Zhou Nie, Yan Huang, and Shouzhuo Yao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03389 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Analytical Chemistry

Multifunctional gold nanoclusters-based nanosurface energy transfer probe for real-time monitoring of cell apoptosis and self-evaluating of pro-apoptotic theranostics Yong Li‡a, Pei Li‡a, b, Rong Zhua, Chao Luoa, Hao Lia, Shanfang Hua, Zhou Niea,* Yan Huanga, and Shouzhuo Yaoa a

State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China b Wuhan Agricultural Inspection Center, Wuhan, 430016, P. R. China * Corresponding author email address: [email protected] (Z. N.); Tel.: +86-731-88821626; Fax: +86-731-88821848

ABSTRACT: Bio-imaging probes for accurately monitoring apoptosis process have extensive significance for cell biological studies and clinical investigations. Herein, a novel multi-functional peptide-tailored gold nanoclusters (AuNCs) has been developed for real-time imaging of caspase-indicated cell apoptosis. The AuNCs nanoprobe was facilely prepared by a one-step peptide-mediated biomineralization with the dye (TRAMA)-tagged peptides specific to caspase 3 as both template agents and the signal switch. Unlike conventional FRET-based fluorescent probes of caspase activity, these nanoprobes relied on the unique quenching effect of AuNCs through the nanosurface energy transfer (NSET) from dye to AuNCs. Intracellular caspase 3 activation cleaved the substrate peptide and released the dye from AuNCs, leading to a significant fluorescence lighting-up for sensitive and continuous analysis of caspase 3 activity in live cells, with a high signal-background ratio, wide linear range (32 pM- 10 nM), and ultralow detection limit (12 pM). Moreover, this versatile AuNCs nanoprobe can serve as a theranostic platform via co-displaying pro-apoptotic and detecting peptides, which allows in situ activation and real-time monitoring of apoptosis in cancer cells. These results indicate that the AuNCs nanoprobe provides a smart molecular imaging and therapeutic agent targeted to cell apoptosis, which has great potential for apoptosis-related diagnosis and precision chemotherapy.

Apoptosis, the most common mode of programmed cell death, is critical for regulation of cell balance and maintaining tissue homeostasis1, 2. Defective apoptosis is a major causative factor of numerous pathological processes, such as autoimmune disorders and cancers3, 4, 5. Accordingly, a potent therapeutic strategy to treat these diseases, particularly in cancer treatment, is to selectively induce cell death through modulating apoptotic pathways3, 6, 7. The detection and monitoring of apoptosis is of considerable significance in not only many cell biological and clinical investigations but also the therapeutic assessment of apoptosis-targeted therapies8, 9. Caspases are a family of cysteine proteases that play an essential role in apoptosis pathways, and its activation has been generally used as the indicator of cell apoptosis10, 11, 12. Unlike traditional apoptosis detection methods, for examples TUNEL assay13 and Annexin V-FITC apoptosis assay14, which are discontinuous and multi-steps, caspase-based assays rely on one-step signal switch caused by caspase-catalyzed peptide cleavage, which are highly suitable for continuous measurement and real-time bio-imaging of apoptosis process. Notably, many intriguing fluorescent nanoprobes, including the dye-labelled gold nanoparticles15, quantum dots (QDs)16, 17, graphene oxide18, 19, and other luminescent nanomaterials20, 21, 22, have been developed as bio-imaging tools for monitoring caspase activity, but the majority of them are single-functional sensor only capable of activity detection. However, multi-functional nanoprobes that enable concurrently inducing and probing of caspase activity in situ or integration of theranostic functions are urgently required by therapeutic monitoring for precision treatment, but their development is still highly challenging. To date, gold nanoclusters (AuNCs), typically consisting of several to tens of gold atoms, have emerged as a new class of nanoprobe for the development of novel sensing methods23, 24, 25 . Due to the excellent photophysical properties, good bio-

compatibility, effective bio-penetration, and feasible surface bio-functionalization, AuNCs-based nanoprobes have been extensively exploited in biosensing26, 27, bioimaging28, 29, 30, and therapeutic applications31. The bio-analytical functions of AuNCs have been successfully proved in metallic ion detection26, biomarker sensing27, protein activity inhibition32 or subcellular organelles bio-imaging28, but their usage in caspase activity assay was scarcely reported. Moreover, it is worth noting that all these AuNCs-based nanosensors are dependent on the fluorescence emission property of AuNCs, besides that some other functionalities of AuNCs have rarely been employed for the sensor development. Recently, Strouse et al discovered that the ultrasmall AuNCs exhibit pronounced energy-absorption capability to quench a luminophore on the surface via nanosurface energy transfer (NSET)33, 34. It is an energy transfer between a molecular dipole and a metal nanosurface that allows the effective distance between nanoquencher and luminophore to surpass the length scare of Foster resonance energy transfer (FRET) pair with traditional dipole–dipole coulombic energy transfer. These findings suggest a new paradigm for the design of AuNCs-based nanosensors using AuNCs as the energy acceptor instead of energy donor. Herein, we reported a novel AuNCs-based nanoprobe for realtime and in situ monitoring of cancer cell apoptosis and noninvasively evaluating theranostics (Scheme 1). In this sensing system, we firstly synthesized peptide-coated AuNCs by a one-step biomineralization method, where the peptides acted as both templates and reducing agents. The resulting AuNCs present efficient broad spectrum quenching properties and can effectively quench the fluorescence of FITC or TAMRAtagged peptides through NSET process. In addition, the peptide decorated on the AuNCs surface retains its intrinsic structure and biological functions which can be hydrolyzed by pro-

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teases and result in a remarkable fluorescence signal-on. Based on the enzyme-trigged fluorescence switch-on, a simple and sensitive fluorescence bioassay was developed for realtime and homogenous detection of caspase 3 (a key apoptotic biomarker) activity and screening its inhibitors. Furthermore, given that AuNCs are good candidate for multi-purpose biofunctionalization, we employed AuNCs as a platform to display both pro-apoptosis peptides and substrate peptides, endowed the nanoprobe with multi-functions of nanocarrier, imaging probe, and apoptosis-inducing drug. This versatile AuNCs-based nanoprobe is able to not only in situ activate and monitor caspase 3-indicated apoptosis in cancer cells, but also real time assess therapeutic efficacy of apoptosis-inducing peptides, holding great potential to be a smart molecular imaging and therapeutic agent for cancer diagnosis and treatment. Scheme 1. The schematic illustration of AuNCs-based nanoprobe for caspase 3-responsive fluorometric biosensor (A), and the schematic diagram of dual-functional AuNCs for in situ activation and bio-imaging of caspase 3-indicated cell apoptosis (B).

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the amount of active protein indicated by the provider (R & D Systems) without calibration. Chloroauric acid (HAuCl4•4H2O), sodium hydroxide (NaOH), sodium chloride(NaCl), ethylene diamine tetraacetic acid (EDTA), trypsin, glutathione (GSH), cysteine (Cys), 2hydroxyethyl (HEPES), and sucrose were purchased from Sigma Aldrich (St. Louis, MO, USA). Staurosporine (STS), dimethylsulfoxide (DMSO) and 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) were obtained from J&K Chemical (Guangzhou, China). HeLa cells were obtained from the cell bank of Central Laboratory at Xiangya Hospital (Changsha, China). RPMI 1640 cell culture medium and fetal bovine serum (FBS) were purchased from Invitrogen (Gibco, USA). Annexin V-FITC apoptosis detection kit, 3-(4, 5-dimethylthiazol-2-yl)-2diphenyltetrazolium bromide (MTT), penicillin-streptomycin, Hoechst 33342 and PBS were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China) and used as received. Phosphate buffer saline (PBS, pH 7.4) containing 136.7 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.47 mM KH2PO4 was used as the bioimaging assay buffer. Caspase 3 assay buffer contained 40 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM EDTA, 10% sucrose, and 0.1% CHAPS. The peptides used in the experiments were chemically synthesized and purified by a solid phase method from KareBay BioChem (Ningbo, China). The sequences and functions of the synthesized peptides are given in Table S1. The primary antibodies caspase 3/p17 Polyclonal Antibody (Rabbit) and β-Tubulin Antibody (Mouse) were purchased from Proteintech (Chicago, IL, USA). The secondary antibodies Goat anti-Rabbit/Mouse IgG(H&L)-HRP were obtained from Bioworld Technology Inc. (Shanghai, China). All other reagents for western blotting were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other reagents and solvents were of analytical grade and used directly. All solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) and had an electric resistance >18.3 MΩ. All the glassware was cleaned with aqua regia (HCl/HNO3 = 3:1, v/v) and thoroughly rinsed with ultrapure water before use.

EXPERIMENT SECTION Chemicals and Materials. Recombinant human caspase 3 protein (active form, 10 µg, 30000 U, one unit is the amount of enzyme that will cleave 1.0 nmol of substrate Ac-DEVD-pNA per hour at 37℃ under saturated substrate concentrations) was purchased from R & D Systems (Minneapolis, USA). Caspase 3 inhibitor Z-DEVD-FMK was purchased from Selleck (USA). Caspase 3 activity assay kit (colorimetric) was obtained from Bioworld Technology Inc. (USA). All concentrations for caspase 3 used in vitro assays were calculated directly using

Preparation and Characterization of Peptide-coated AuNCs. In a typical experiment, 16 µL freshly prepared aqueous solution of HAuCl4 (25 mM) was slowly added to 376 µL aqueous solution of peptides (containing dual peptides with different concentration ratio, total final concentration to be 1 mM) in a 1.5 mL Eppendorf tube with vigorous stirring. Then 8 µL 1 M NaOH was added within 30 seconds to adjust the solution pH approaching about 10. The sample was stored and sealed for 12 h in the dark undisturbedly to produce the peptide-coated AuNCs. The resulting AuNCs (400 µL) were concentrated and washed with ultrapure water for three times by ultrafiltration using a centrifugal filter unit (Amicon Ultra-0.5 mL with MWCO of 10000, Millipore) so as to remove excessive peptide and HAuCl4. After that, the concentrated AuNCs were diluted with ultrapure water to 400 µL. At last, the final synthesized AuNCs were stored at 4 oC, certain volume of AuNCs was taken out and the volume concentration of AuNCs


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were fixed as the quantification required. All the synthesized AuNCs was listed in Table S2 of Supporting Information part.

cation Suite Advanced Fluorescence (LAS-AF) software package.

In Vitro Detection of Caspase 3 Activity. In a typical caspase 3 activity assay, 2 µL AuNCs (30 nM) was added into 88 µL caspase 3 reaction buffer (containing 40 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM EDTA, 10% sucrose, and 0.1% CHAPS), then 10 µL caspase 3 (final concentrations ranging from 0 to 10 nM) or other proteins (200 nM) was added and incubated at 37 °C for the designed time. After reaction, the resulting solution was subjected to fluorescence measurements (QuantaMasterTM4 fluorescence spectrophotometer, PTI, Canada). The fluorescence spectra were collected with both excitation and emission slits of 5 nm, for TAMRA the scanning range was from 560 to 700 nm under an excitation of 550 nm and for FITC the scanning range was from 490 to 650 nm under an excitation at 480 nm, respectively. The timedependent fluorescence responses were recoded immediately after the addition of caspase 3 at an excitation wavelength of 550 nm (slit 5 nm) and an emission wavelength of 580 nm (slit 5 nm) with a time interval of 8 s.

Please see Supporting Information (SI) for more experimental details, such as instrumentation and characterization, measurement of the fluorescence quantum yields of AuNCs, calculate the number of peptides on AuNCs, calculation of the AuNCs concentrainon, cell culture, caspase 3 activity assay in cell extracts, western blotting (WB) analysis, caspase 3 detection by flow cytometry, cytotoxicity assay, and statistics analysis.

Apoptosis Analysis by Flow Cytometry. The apoptosis processes of HeLa cells were evaluated by Annexin V-FITC apoptosis detection kit. HeLa cells were seeded in the 12-well plates at a density of 5 × 104 cells and containing 1 mL fresh RPMI-1640 per well. After incubated for 24 h, peptides (2 µM), STS (4 µM), and kinds of AuNCs (6 µL, 30 nM) were added to 300 µL solution and then co-incubated for another 3 h, respectively. Then the resulting cells were washed with PBS for three times, digested by trypsin (EDTA deplete), collected by centrifugation (2000 rpm for 5 min). After washing with PBS for two times again, the cells were re-suspended in 0.5 mL binding buffer. Thereafter, 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) were added to each centrifuge tube with thoroughly mixing. After incubating in dark at room temperature for 10 min, the cell samples were analyzed by flow cytometry (Gallios flow cytometer, Beckman-Coulte, USA) over FL1 (Annexin V-FITC) and FL3 (PI) channels. Confocal Laser Scanning Microscopic (CLSM) Imaging of Living Cells Apoptosis. The apoptotic fluorescence of HeLa cells was visualized with a confocal laser scanning microscope. HeLa cells (2 × 104 cells per dish) were seeded into confocal dishes with 10 mm bottom well in RPMI 1640 medium for 24 h. When the cells were about 80% confluent, the coverslips were washed three times with PBS, then the medium was replaced with fresh culture medium containing different functionalized AuNCs probe (6 µL, 30 nM) or STS (4 µM) and incubated at 37 °C for particular time. If necessary, caspase 3 inhibitor (100 µM Z-DEVD-FMK) was pre-incubated with HeLa cells for 1 h before the addition of AuNCs. The incubating time for AuNCs was 3 h for cell uptake of the nanoassembly in their separate culture medium. The media were removed and each well was washed twice with cold PBS to remove the residual agents. Thereafter, all the nuclei were stained with 5.0 µg mL-1 Hoechst 33342 for 10 min at 37 °C, and the cells were further washed with PBS for three times before imaging. Subsequently, the cells were visualized on a confocal laser scanning microscope (CLSM, C1-Si, Nikon, Japan). Hoechst 33342 was excited with a 405 nm laser diode and the emission was collected from 420 to 480 nm. The fluorescence of released TAMRA was collected from 580 to 620 nm on the microscope with the excitation wavelength of 565 nm. All images were digitized and analyzed with Leica Appli-

RESULTS AND DISCUSSION Synthesis, Characterization, and Optimization of the AuNCs Nanoprobes. The peptide-functionalized AuNCs nanoprobes were synthesized according to a reported method with specific modifications28. In our synthesis method, two peptides, the capping peptide Pc (GGGGGLVPEGSCCYNH2) and the probe peptide Pp (CCYGGGGRGRK (FITC)GNH2), were utilized as reductant and co-template for the formation of AuNCs. Both the sequences of two peptides were rationally designed and shared the same reducing domain CCY, in which the phenolic group of tyrosine (Y) under alkaline conditions (pH=10) is able to reduce Au3+ to Au0 and the cysteine residues were served as the anchoring groups to bind with AuNCs via thiol-gold interaction. Besides the reducing domain (CCY), the probe peptide was also composed of enzyme-substrate domain labelled by fluorophore tag, and in our first testing case the specific substrate sequence of trypsin, a typical protease, and FITC tag were used as the model system.

Figure 1. The characterization of peptide-coated AuNCs, including the absorption spectrum (A), the fluorescent spectrum (B), and the transmission electron microscopy (TEM), left panel: the TEM of the distribution, and the lattice fringes (inset image) of AuNCs. Right panel: the magnified HR-TEM (1), temperature colored HRTEM (2), fast Fourier transform (FFT) spectrum (3) and inverse fast Fourier transform (IFFT) TEM images (4) of squared area in left panel (C). The diameter distribution of AuNCs by statistical calculation of 150 particles (D). The XPS spectra of Au 4f (E) and S 2p for peptide-AuNCs (F).



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At first, the AuNCs nanoprobes were prepared by mixing HAuCl4 with peptides under alkaline conditions (pH=10) in the dark for 12 h, which was a facile one-pot green biomineralization process without requiring any strong reducing agents. After simple centrifugal purification, the resulting AuNCs nanoprobes (PpPc@AuNCs) were obtained and then well characterized. The UV-vis spectum of AuNCs probes shows a broad absorption band with two weak shoulders at 239 nm and 294 nm, which are attributed to the phenoxide structure of tyrosine (Y) in peptides28, 35, indicating that the oxidation of tyrosine is responsible for gold ion reduction (Figure 1A). Also, besides an obvious absorption peak at 490 nm belonging to FITC, a gradient weak absorption from 350 to 800 nm was observed which is consistent with the previously reported results of AuNCs36, 37, 38, 39. Meanwhile, the fluorescence emission spectra of AuNCs probes exhibit two interesting phenomena: (1) the emission at 515 nm of the FITC-tagged PpPC@AuNCs probes is markedly weaker than that of FITCPp with equal equivalent concentration of peptide, strongly proving the efficient quenching of FITC by the AuNCs (Figure 1B); (2) unlike the previously reported peptide-protected AuNCs with red emission, our AuNCs probes possess negligible fluorescence emission at 600 nm, which is also confirmed by the control PpPc@AuNC without FITC-labeling (Figure 1B, blue line), probably due to the alternation of peptide sequences which is a key factor for peptide-mediated metal nanocluster bio-mineralization38, 39. A typical TEM image reflects that the as-prepared peptide-protected AuNCs are well monodispersed and uniformed in size with an average diameter of around 1.7 nm (Figure 1C and Figure 1D). The highresolution transmission electron microscope (HR-TEM) coordinating with the temperature colored HRTEM, fast Fourier transform (FFT) spectrum and inverse fast Fourier transform (IFFT) TEM show that their lattice fringes are ∼0.23 nm (right panel of Figure 1C), which is consistent with the interplanar spacing of the (111) crystal plane of face-centered cubic Au29. Additionally, it was observed from the AFM image that the topographic height of peptide-protected AuNCs is about 2 nm (Figure S1) that is in accordance with the TEM data. The indepth chemical states of the PpPc@AuNCs were further characterized by X-ray photoelectron spectroscopy (XPS). Two intense peaks were obtained at 83.4 and 87.2 eV, which were assigned to the 4f7/2 and 4f5/2 features of Au (0)27, 40 (Figure 1E). Moreover, the S 2p3/2 peak (Figure 1F) at 161.9 eV confirmed the existence of Au-S bond which helps peptide locating on the surface of AuNCs as literature noted27. All these results validate the successful preparation of the peptideprotected AuNCs with good-dispensability and great quenching property. For further investigating the quenching effect of peptidefunctionalized AuNCs, another red fluorochrome of TAMRA and its corresponding AuNCs capped by TAMRA-Pp (CCYGGGGRGRK(TRAMA)G-NH2) and Pc were introduced for comparison. The detailed analysis of fluorescence quantum yield (QY) of fluorescent dye-tagged peptides demonstrated that after AuNCs generation, the QY of FITC decreased from 0.41 to 0.07 and the QY of TAMRA decreased more sharply from 0.63 to 0.05, suggesting that the AuNCs have a broad spectrum quenching ability to quench the fluorescence of both FITC and TAMRA which is speculated through the nanosurface energy transfer (NSET) mechanism33, 34. In the control experiments with the mixtures of FITC or TAMRA monomers and Pc-coated AuNCs, negligible fluorescence quenching of

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FITC or TAMRA monomers was observed (Figure S2), implying that the proximity of fluorophores to the surface of AuNCs by peptide anchoring is essential for the high-efficient quenching. In fact, due to the broad absorption ability, AuNCs absorbed the fluorescence emitted by fluorochromes nearby AuNCs, then the energy was transferred to the metal surface of AuNCs with loss of energy via heat33, 34, 41. It is worth noting that AuNCs show higher quenching efficiency on redemissive dye of TAMRA than green-emissive dye of FITC, which is consistent with the reported fact that lower energy dye has an enhanced efficiency of energy transfer over the higher energy dye in NSET process33, 34.

Figure 2. Normalized fluorescence spectra of Trypsin-responsive PpPc@AuNCs labelled with fluorochrome of FITC (A) and TRAMA (B), respectively. The dot curve of fluorescence changes of the PpPc@AuNCs upon treatment with thiol-containing molecules or cell lysis for different durations (C) and their response to trypsin after storage for different days (D).

In order to optimize the protease-responsive performance of our PpPc@AuNCs nanoprobes, different proportions of fluorescence dyes-labelled Pp to Pc in the synthetic precursors of AuNCs were investigated, and the fluorescent signal-tobackground ratios (S/B) of their products in response to protease cleavage were evaluated. Since the Pp in these testing samples includes the specific recognition sites of trypsin (arginine(R) and lysine (K)), once 20 nM trypsin was added into AuNCs solution (containing 20 µM synthesized peptide), significant fluorescence recovery was observed in both FITC and TAMRA-labeled AuNCs probes (Figure 2A and 2B). The data of optimization experiments show that the best ratio of Pp: Pc in response to trypsin for FITC-labeled AuNCs probes was 1:30 with the maximal signal-to-background ratio(S/B) of 6.8 (Figure S3), whereas the nanoprobes tagged with TAMRA have the best S/B of 19.7 (Figure 2B) also under the optimal ratio of Pp: Pc to be 1: 30 (Figure S4). The better responsive performance of TAMRA-labeling compared to FITC is probably due to its higher efficiency of NSET, suggesting that TAMRA is preferable to our NSET-based AuNCs nanoprobes, and thus TAMRA-labeled AuNC nanoprobes prepared upon the Pp: Pc ratio of 1: 30 was exploited in the following detective experiments. The stability and anti-interference of the obtained nanoprobes were also thoroughly examined. Their emission spectra show negligible changes not only upon storage at room temperature for 30 days (Figure 2C, black dot), but also in the presence of different thiol-containing biomolecules (GSH, Cys and RCG peptide), and even cell lysate.


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Moreover, their enzyme-specific response was also unimpaired within 30 days (Figure 2D, red dot). All these data implies that the peptide-protected AuNCs with high quenching ability possess superb stability, strong anti-interference property, and excellent biological response. In Vitro Detection and Living Cells Imaging of Caspase 3 Activity using Caspase 3-specific AuNC Probes. To demonstrate the validity of our protease-responsive AuNCs nanoprobes for caspase detection, we prepared a novel caspasespecific AuNCs probes for fluorescent assaying and bioimaging of caspase activity. Caspase 3 is a key member of caspase family and plays the role as the downstream executioner to bring about the irreversible processing of cell apoptosis. Herein, the caspase 3 probe peptide (Pcp, TAMRAGGDEVDGGRGGRGGCCY-NH2), which comprises the recognition site (DEVD) for caspase 3 and TAMRA label, and Pc were selected as co-template for the synthesis of caspase 3 responsive AuNCs (PcpPc@AuNCs). According to the aboveoptimized condition, AuNCs was synthesized under the optimal condition with the molar ratio of Pcp/Pp of 1:30. As anticipated, the PcpPc@AuNCs probe retains fluorescence off state due to the high efficient NSET from TAMRA to AuNCs surface (Figure 3A, black line), whereas the addition of 10 nM caspase 3 significantly increased the fluorescence signal by 18.4-fold (red line of Figure 3A), indicating that caspase 3 specifically cleaved the substrate peptide of Pcp and efficiently released TAMRA from AuNCs. Based on the remarkable fluorescence turn-on of caspase 3 responsive to PcpPc@AuNCs, quantitative analysis of caspase 3 activity could be achieved. Figure 3B revealed that the fluorescence signal increased as a function of the concentration of caspase 3, and its calibration curve has a linear range from 32 pM (0.0972 U/mL) to 10 nM (30.37 U/mL) (r2=0.993) with a detection limit (3σ) of 12 pM (0.0365 U/mL), which is much more sensitive than other previously reported methods42, 43, 44, 45, 46, illustrated in Table S3. We next exploited the AuNCs-based nanoprobe to dynamically monitor caspase 3 activity in real time. The fluorescence emission at 580 nm was continuously measured from 0 min once 2.5 nM caspase 3 was added. Figure S5 depicts the typical real-time fluorescence curves, which exhibits that the fluorescence intensity steeply increased from 0 to 0.5 h, then shifted to slow increase and almost reached the saturation. This result confirmed that our AuNCs-based nanoprobe is feasible for continuously monitoring caspase 3 activity and is practical for real-time imaging of the caspase-indicated cell apoptosis. Furthermore, the specificity of the nanoprobes was evaluated by challenging other interferent proteins or enzymes, including hemoglobin, BSA, lysozyme, thrombin, sortase, alkaline phosphatase (ALP) and carboxypeptidase (CPY). The reaction system did not display any appreciable fluorescence activation even though that the interferent proteins were 20 times the concentration of caspase 3 (Figure 3C), validating the excellent selectivity of our sensor. Owing to that caspase inhibitors are potential clinical agents for the treatment of various diseases, including neurological disorder and lung disease, the feasibility of the AuNCs-based nanoprobe in screening of caspase 3-targeted inhibitors was also assessed. Taking ZDEVD-FMK as an example inhibitor, the inhibitor concentration-dependent fluorescence decrease was observed in this system (Figure S6), and it gave an IC50 to be about 1.21 µM,

proving that our method is applicable to caspase-targeted inhibitor screening.

Figure 3. Fluorescence spectra of PcpPc@AuNCs in the presence and absence of 10 nM caspase 3 (A). Changes in fluorescent emission spectra after addition of different concentrations of caspase 3 and the linearity curve (inset) corresponding to fluorescent intensity at 580 nm versus concentration of caspase 3 increasing from 0 to 10 nM (B). The bar graph of fluorescent changes of PcpPc@AuNCs probe responsive to various kinds of enzymes, including 200 nM hemoglobin, BSA, lysozyme, thrombin, sortase, alkaline phosphatase (ALP) and carboxypeptidase (CPY), and 10 nM caspase 3 (C). The bar graph of fluorescent changes of PcpPc@AuNCs probe responsive to non-apoptotic HeLa cell lysis and apoptotic HeLa cell lysis.

Prior to the usage of the AuNCs-based nanoprobe in caspase activity analysis in cell lysate and living cells, we found that the fluorescence responses of the AuNCs to 2.5 nM caspase 3 remained unchanged in the presence of cell culture media and the non-apoptotic HeLa cells lysis (Figure S7), proving the robustness of our sensor in complex cell culture matrix. Next, the activity of caspase 3 was monitored in the cell lysate of drug-treated apoptosis cells. HeLa cells (50000 and 100000 numbers HeLa cells, respectively) were treated by a typical apoptosis chemo-inducer (staurosporine, STS, 4 µM), and then their lysates were collected. Incubation of the lysates with the nanoprobes caused substantial increase of fluorescence at 580 nm (Figure 3D), and the obtained concentrations of caspase 3 were comparable to those of caspase 3 assay kit (Figure S8), suggesting the intracellular activation of caspase 3 induced by STS. Considering the importance of real-time and in-situ tracking of cell apoptosis in cell biology research, we further employ our AuNCs-based nanoprobe to monitor the activity of caspase 3 in living cells. Confocal laser scanning microscopy (CLSM) was used to visualize the intracellular activation of caspase 3. Figure 4 shows representative cellular images of non-apoptotic HeLa cells and apoptotic HeLa cells after incubating with PcpPc@AuNCs (containing about 60 µM synthesized peptide). It was found that strong red fluorescence signals are collected from the STS-induced apoptotic HeLa cells with PcpPc@AuNCs treatment (middle row). In sharp contrast, little red fluorescence of TRAMA was emitted in the normal HeLa cell without pretreating apoptotic agent of 4 µM STS (first row). Moreover, once caspase 3 inhibitor (100 µM Z-DEVD-FMK) was pretreated into the apoptotic HeLa cells, then following the addition of PcpPc@AuNCs, the red fluorescence is sharply weakened (third row), indicative of the activity of caspase 3 greatly inhibited. Furthermore, all cells



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were co-stained with Hoechst 33342, which binds strongly to DNA and forms a blue fluorescent complex in nuclei of cell47. As presented in the merged images, blue images are all explicitly shown in the nucleus area, whereas the TRAMA-emitted red fluorescence was merely distributed in the cytosol area nearby nuclei. This is because that caspase cascade is mostly activated in cytoplasm during the STS-induced mitochondria apoptosis pathway48. In all, the above results revealed a direct correlation between intracellular caspase 3-activated fluorescence imaging and STS-induced apoptosis.

Figure 4. Confocal laser scanning microscopy (CLSM) images of HeLa cells treated with caspase 3-responsive nanoprobe of PcpPc@AuNCs (6 µL, row 1), PcpPc@AuNCs in the presence of 4 µM STS (row 2), and PcpPc@AuNCs in the presence of 4 µM STS and 100 µM Z-DEVD-FMK (row 3), and the scale bar is 10 µm.

In Situ Activation and Bio-imaging of Caspase 3 by Proapoptotic/Detecting Dual-functional AuNCs. Our first version of caspase-responsive AuNCs nanoprobes still requires additional inducer to trigger the activation of apoptosis, whereas integration of pro-apoptotic and detecting functions into one nanoprobe will provide a more convenient toolkit for in situ induction and noninvasive monitoring of caspaseindicated apoptosis in living cells. Moreover, this dualfunctional nanomaterial is also a highly promising platform for theranostic applications because it works as both the clinical agent targeted to cell apoptosis and the indicators for real-time assessing the therapeutic efficiency. Hence, it is of importance to develop dual-functional nanoprobes capable of proapoptosis and detecting. Our peptide-functionalized AuNCs whose surface displays two types of peptides can perfectly address this goal because there is considerable versatility via simply changing the peptide sequences with different functions. Recently, increasing number of polypeptide theranostic drugs, especially apoptosis-inducing peptides, were exploited for biomedical applications such as traceable cancer therapy49, 50 . One representative pro-apoptotic peptide KLA is a mitochondria-targeted therapeutic agent, which can induce the disruption of mitochondrial membranes, mitochondrial swelling, and sequentially mitochondria-dependent cell apoptosis51, 52 . However, it has poor cell penetrating potentials or cellular penetrating ability for its low positive charged potential, thus the integration of this peptide with nanocarriers would be highly desirable for improving its efficiency. Herein, we rationally designed an apoptosis-inducing peptide (Pa) coating on AuNCs surface (Scheme 1B), which comprises three domains: (1) a pro-apoptotic domain containing KLA sequences; (2) a cancer cell-targeting domain with RGD sequence for specific tumor targeting and assisting cell internalization, since

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RGD motif can specifically bind to Integrin αvβ3 which is highly expressed in the tumor cells53; (3) a AuNCs-reduction and capping domain (CCY). Taking advantage of Pa and cooperating with caspase 3 substrate peptide of Pcp and Pc for AuNCs synthesis, we successfully prepared a proapoptotic/detecting dual-functional nanoprobe of PcpPa@AuNCs, and also prepared a series of mono-functional control AuNCs for comparison, including Pa@AuNCs (proapoptosis only), PcpPc@AuNCs (detection only), and Pc@AuNCs (control). At first, the apoptosis-inducing ability of pro-apoptotic peptide-functionalized AuNCs was examined. The cytotoxicity of peptides and peptide-coated AuNCs was evaluated using the standard MTT assay. After incubating HeLa cells with kinds of peptides or AuNCs, only pro-apoptosis peptide synthesized AuNCs (Pa@AuNCs and PcpPa@AuNCs) seriously impeded cell proliferation with a low relative cell viability of 15% in a short period of 4 h, and the apoptotic peptide showed a partly cytotoxicity of about 70% cell viability after 24 h, whereas the other peptides and AuNCs showed negligible effect on cell viability after 24 h incubation (Figure S9). Annexin VFITC/PI double staining assay with flow cytometry detection was further implemented to evaluate the extent of cell apoptosis caused by peptides and AuNCs. As shown in Figure 5, both pro-apoptotic AuNCs (6 µL Pa@AuNCs or PcpPa@AuNCs, containing an approximate concentration of 2 µM Pa) induced notable apoptosis of HeLa cells in 3 h with the early apoptotic rate of 77.01 % and 68.73 %, respectively, which is comparable with that of 4 µM typical chemo-inducer STS (77.05 %). Interestingly, the pro-apoptotic peptide Pa (2 µM, an approximate concentration as Pa in AuNCs) led to relatively small apoptosis ratio (23.63 %) after incubating with HeLa cells for 24 h, implying that pro-apoptotic peptide loaded on the nanocarrier of AuNCs could improve its therapeutic efficiency. As expected, other peptides and AuNCs without pro-apoptotic function show little apoptosis (apoptotic rate < 6 %) compared with control sample (apoptotic rate 1.7 %).

Figure 5. Flow cytometric analysis of the HeLa cells (A) and HeLa cells treated with 2 µM Pcp for 3 h (B), 2 µM Pa for 3 h (C), 2 µM Pa for 24 h (D), 6 µL PcpPc@AuNCs for 3 h (E), 6 µL Pa@AuNCs for 3 h (F), 6 µL PcpPa@AuNCs (without TAMRA labeling) for 3 h (G), and 4 µM STS for 3 h (H) using apoptosis assay kit with the dual fluorescence of Annexin V-FITC/PI double staining.

Furthermore, detailed research showed that Pa@AuNCs could induce cell apoptosis in a time-dependent manner (Figure S10), and the early apoptotic ratio increased significantly as treating time increasing from 0.5 to 4 h. In addition, the apoptotic result induced by PcpPa@AuNCs was also supported by confocal laser scanning microscopic (CLSM) imaging through


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the double-staining of Annexin V-FITC/PI (Figure S11). The HeLa cells exposed to STS or PcpPa@AuNCs revealed evident green fluorescence of Annexin V-FITC-stained cell membrane and strong red fluorescence in the PI-stained nucleus. In contrast, neither green nor red fluorescence was observed in cell lines pre-incubated with non-apoptotic PcpPc@AuNCs. Overall, all these results demonstrated that the coated pro-apoptotic peptide on AuNCs retained and even improved its cell-toxic functions, and AuNCs provided a good scaffold for displaying therapeutic peptides to exert anticancer activity by inducing cell apoptosis. The proposed dual-functional AuNCs nanoprobe can be developed to a therapeutic platform acting as a nano-sensor and nano-drug. This “smart” platform is able to specifically target and efficiently penetrate into cancer cells, activating apoptosis within a short period of time, and consequently its built-in apoptosis sensor can in situ report the therapy efficacy at a very early stage. To validate these features, CLSM imaging was employed to visualize the nanoprobes-caused cell apoptosis and its caspase-responsive fluorescence signals. All cells were co-treated with Hoechst 33342 for nuclei staining. The detection-only nanoprobes, PcpPc@AuNCs, in the absence of exogenous apoptosis inducer shows no emission in the red channel as noted in Figure 6A (row 1), indicative of little cell apoptosis as well as caspase 3 activity. In sharp contrast as Figure 6A (row 2) show, cells treated by the dual-functional PcpPa@AuNCs underwent severe apoptosis showing shrinkage, budding, and apoptotic body formation, demonstrating maximum TAMRA fluorescence signals resulted from caspase 3 activation and cleavage. Meanwhile, caspase 3 inhibitor ZDEVD-FMK could greatly reduce the fluorescence signal (row 3), validating that the fluorescence response of the nanosensor was specific to caspase 3-activated apoptosis. Therefore, it evidenced the novel PcpPa@AuNCs-based probe could be used to monitor therapeutic efficacy.

Figure 6. The CLSM images of HeLa cells treated with 6 µL mono-functional PcpPc@AuNCs (row 1), and 6 µL dualfunctional PcpPa@AuNCs in the absence (row 2) and presence (row 3) of 100 µM Z-DEVD-FMK (A), and the scale bar is 10 µm. The standard western blotting assay of caspase 3 with HeLa cells treated by no probe (1), 6 µL PcpPc@AuNCs (2), 6 µL PcpPa@AuNCs (3), 6 µL Pa@AuNCs (4), and 4 µM STS (5) (B). The curve of flow cytometer analysis captured by fluorescence of TRAMA with HeLa cells treated by no probe (grey curve), 6 µL PcpPc@AuNCs (green curve), 6 µL PcpPa@AuNCs (amaranthine curve), and 6 µL PcpPc@AuNCs after addition of 4 µM STS (salmon curve) (C).

In addition, the PcpPa@AuNCs-induced caspase 3 activation was further confirmed by (1) probing caspase 3 activity in extracts of the corresponding batch of cells using a colorimetric caspase 3 assay kit (Figure S12) and (2) analyzing the cleavage of procaspase 3 using a standard western blotting assay (Figure 6B). Then, the caspase 3 activation-caused fluo-

rescence signal was quantitatively investigated by flow cytometers analysis through monitoring the fluorescence of TRAMA released from the self-reporting nanoprobes of PcpPa@AuNCs (Figure 6C). In comparison with the control or PcpPc@AuNCs-treated sample, HeLa cells treated by PcpPa@AuNCs show about 20-fold fluorescence increase. Hence, it is more accurately reliable that PcpPa@AuNCs possess both the cell-apoptosis induction and detection capability in living cancer cells, which could serve as a caspase-targeted theranostic probe for treating cancer. To further analyze the time-dependent therapeutic process of PcpPa@AuNCs, the CLSM images of HeLa cells were acquired after different incubation time intervals (Figure 7A). Only weak red fluorescence was observed after 30 min. However, the fluorescence intensity was remarkably enhanced with gradually obvious shrinking morphology of cells at prolonged incubation time, indicating the progressive caspase activation and the processing of apoptosis. These results clearly demonstrate that PcpPa@AuNCs not only can be used for real-time detection of caspase 3 activity and cell apoptosis but also have the potential for in situ reporting the peptide-directed therapeutic response at single cell level. Additionally, cell apoptosis induced by pro-apoptotic AuNCs shows a dosage-dependent manner, which can also be monitored by its fluorescent selfreporting property (Figure S13). Moreover, the cell-apoptosis imaging ability of PcpPa@AuNCs was further verified, and Zscanning confocal imaging was performed to visualize the intracellular fluorescence distributions in HeLa cells (Figure 7B). It is apparent that bright red fluorescence existed throughout whole cytoplasm, which suggested an efficient delivery of the AuNCs nanoprobes and the AuNCs nanosensing platform afforded a robust intracellular caspase 3 activity sensor for high-contrast and continuous imaging during the apoptosis-induced therapeutic process.

Figure 7. Real-time fluorescence imaging of of HeLa cells treated with 6 µL PcpPa@AuNCs (A), and the Z-scanning confocal laser scanning microscopy of apoptotic HeLa cells capturing from 0 µm to 22 µm (B). The scale bar is 10µm.

CONCLUSION In this proof-of-concept work, a novel fluorescence lightingup probe for detection and bio-imaging of caspase 3 activity has been developed based on appealing NSET-dependent quenching effect of the peptide-coated ultra-small AuNCs. Moreover, by one-step functionalization of AuNCs with both pro-apoptotic peptide and caspase 3 specific probe peptide, a multifunctional AuNCs probe capable of in situ apoptosis in-



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duction and simultaneous processing evaluation has been fabricated for potential anti-cancer theranostic applications. The proposed nanoprobes are generally applicable for in vitro enzymatic activity assay, flow cytometric analysis, and living cells bioimaging. Compared with the conventional molecular or nanoprobes that can only monitor the activity of caspase 3, this nanosensor demonstrates some inherent advantages as following: (1) for cell biological research, it is ultra-small (1-2 nm) with high cell-penetrating ability and less interference on cell functions; (2) its preparation integrates synthesis and functional modification in one step, which is simple, green, lowcost, and highly versatile for multi-functionalization; (3) it allows high contrast imaging with good signal-to-background ratio; (4) it provides extra therapeutic function via welldefined apoptosis-targeted pathway. We envisage that the proposed AuNCs probes will be able to present a novel design concept of apoptosis biosensors and theranostic agents, which might be promising for apoptosis-targeted drug discovery and cancer precision chemotherapy.

ASSOCIATED CONTENT Supporting Information Please see Supporting Information (SI) for more experimental details and other tables (Table S1-S3) or figures (Figure S1-S13). The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Corresponding author. Tel.: +86-731-88821626; Fax: +86-73188821848; E-mail address: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Yong Li and Pei Li contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Nos. 21235002, 21575038, and 21475037), the Foundation for Innovative Research Groups of NSFC (Grant 21521063), Young Top-notch Talent for Ten Thousand Talent Program, the Natural Science Foundation of Hunan Province (No. 2015JJ1005).

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for TOC only


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