Multi-FÃ¶rster Resonance Energy Transfer-Based Fluorescent Probe

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Letter

A multi-FRET-based fluorescent probe for spatiotemporal MMP-2 and caspase-3 imaging Hong Cheng, Shi-Ying Li, Hao-Ran Zheng, Chu-Xin Li, Bo-Ru Xie, Ke-Wei Chen, Bin Li, and Xianzheng Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00277 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Analytical Chemistry

A multi-FRET-based fluorescent probe for spatiotemporal MMP-2 and caspase-3 imaging Hong Cheng,† Shi-Ying Li,† Hao-Ran Zheng,† Chu-Xin Li,† Bo-Ru Xie,† Ke-Wei Chen,† Bin Li,† Xian-Zheng Zhang*,† †

Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, The Institute for Advanced Studies, Wuhan University, Wuhan 430072, P. R. China * Fax: +86 27 6875 4509. E-mail: [email protected] ABSTRACT: A novel single-molecular fluorescent probe (short as Mc-Probe) was developed for spatiotemporal MMP-2 and caspase-3 imaging with distinct fluorescence signals. Due to the multi-FRET processes, Mc-Probe could respond to MMP-2 and caspase-3 independently with high signal-to-noise ratio. Moreover, the overexpression of MMP-2 in cancer cell lines and the cisplatin induced cell apoptosis was spatiotemporal imaged with distinct fluorescence emissions. Because of the independent process of Mc-Probe for MMP-2 and caspase-3 imaging, Mc-Probe could meet the demands for precise disease diagnosis and cancer theranostic applications, which could extensively simplify the processes for precise cancer diagnosis and imaging.

INTRODUCTION The cancer progressions are regulated by the spatiotemporal distributed enzymes.1,2 For example, an increased Matrix metalloproteinases (MMPs) expression was observed in almost all human cancers, which could promote cancer angiogenesis, invasion and metastasis.3-5 Besides, the elevated activities of caspases could induce some severe pathologies, which also played a vital role in programmed cell death.6-8 In the past decades, MMP-2 and caspase-3 were the mostly used biomarkers for cancer diagnosis, therapeutic drug screening and cancer theranostics.9-14 To date, a variety of fluorescent probes had been developed for MMP-2 or caspase-3 imaging with high selectivity and sensitivity.15-19 However, it was often difficult to integrate them together excellently due to their different localization and metabolisms, which was also unsuitable for dynamic intracellular signal transduction monitoring.20-23 Thus, a novel fluorescent probe was urgently needed for simultaneously MMP-2 and caspase-3 imaging. Fӧrster resonance energy transfer (FRET) is a unique approach for noninvasive biological fluorescence imaging.24-27 Nevertheless, the energy transfer efficiency was greatly limited by the inherent characteristics of the fluorophores and the conformation diversity of the linkers, while the unquenched fluorescence would enhance the unwanted background signal.28,29 A higher fluorescence quenching efficacy was preferred to enhance the signal-to-

noise ratio for practical applications. An alternative strategy for improving the FRET efficiency was applied by introducing multiple quenchers into the probe to increase the absorbtion efficiency and enhance the dipole-dipole coupling interactions between fluorophores.30,31 Even so, the single emission phenomenon could not be applied in spatiotemporal multi-object imaging distinctively. Here, we designed and characterized a multi-FRETbased fluorescent probe for spatiotemporal MMP-2 and caspase-3 imaging with distinct fluorescence emissions. As shown in Scheme 1, the probe of FAMSDEVDSK(TAMRA)GPLGVRGK(Dabcyl) (short as McProbe) was comprised of a MMP-2 sensitive peptide linker GPLG*VRG (“*” representing the cleavage site), a caspase-3 specifically responsive peptide SDEVD/S (“/” representing the cleavage site), and FAM/TAMRA/Dabcyl based multiFRET donor-acceptor fluorophores. Among which, FAM was the donor for both TAMRA and Dabcyl. Dabcyl was the acceptor for both FAM and TAMRA. TAMRA was the donor in TAMRA/Dabcyl FRET process, and it also acted as the acceptor in FAM/TAMRA FRET pair. Both FAM and TAMRA fluorescence were originally quenched by FRET processes. After recognized and cleaved by MMP-2, the changes in the TAMRA/Dabcyl separation distance would induce the termination of the FRET process with the fluorescence recovery of TAMRA. Subsequently, the activated caspase-3 could further cleave the probe, change the sepa-

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ration distance between FAM and TAMRA and finally terminate the FRET process with the fluorescence recovery of

Scheme 1. Chemical structure and the proposed processes of Mc-Probe for spatiotemporal MMP-2 and caspase-3 imaging. FAM. This multi-FRET-based fluorescent imaging could be used for precise disease diagnosis or therapeutic efficacy evaluation.

EXPERIMENTAL SECTION Materials and Methods. Rink Amide resin (100–200 mesh, loading: 0.59 mmol/g), N-Fluorenyl-9methoxycarbonyl (FMOC) protected L-amino acids (FMOC-Lys(Mtt)-OH, FMOC-Ser(tBu)-OH, FMOCAsp(tBu)-OH, FMOC-Glu(tBu)-OH, FMOC-Val-OH, FMOC-Gly-OH, FMOC-Leu-OH, FMOC-Pro-OH and FMOC-Arg(pbf)-OH), 1-hydroxybenzotriazole (HOBt), obenzotriazole-N,N,N’,N’tetramethyluroniumhexafluorophosphate (HBTU), and Diisopropylethylamine (DIEA) were purchased from GL Biochem. Ltd. (Shanghai, China). Piperidine, N,N’dimethylformamide (DMF), methanol, dichloromethane (DCM) and anhydrous ether were obtained from Shanghai Chemical Co. (China). DMF was used after distillation. Trifluoroacetic acid (TFA), 1,10-phenanthroline monohydrate, cisplatin, acetic anhydride, 5(6)-Carboxyfluorescein (FAM) and 2,6-lutidine were obtained from Aladdin Reagent Co. Ltd. (Shanghai, China). 5(6)Carboxytetramethylrhodamine hydrochloride was purchased from Heowns corp. (Tianjing, China). N-

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methylmorpholine (NMM), 4-{[4-(Dimethylamino)phenyl]-azo}-benzoic acid (Dabcyl) were obtained from TCI corp. (Shanghai, China). Staurosporine, caspase-3 (human recombinant, Lot No. 9B241083-6) and caspase-9 (human recombinant, Lot No. 81201089) were purchased from Biovision corp.. Ac-DEVD-CHO and Matrix metalloproteinases (MMP-2, catalog: 902-MP-010) were purchased from R&D Systems. Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (PBS), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium-bromide (MTT), fetal bovine serum (FBS) were purchased from Invitrogen Corp., All other reagents were of analytical grade and used as received. High performance liquid chromatography (HPLC) was used to analysis of the purity of the probe. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin–Elmer). The electrospray ionization-mass spectrometry (ESI-MS) was used to confirm the structure of the probe. Fluorescence microscopy images were observed using a confocal laser scanning microscopy (C1-Si, Nikon, Japan). The absorbance of Dabcyl, TAMRA and PBS were recorded on a UV-vis spectroscopy (Lambda Bio40). Quantitative fluorescence was analyzed by flow cytometry (BD FACS Aria TM III). Cell Cultures. African green monkey fibroblast (COS7) cells and Squamous cell carcinoma (SCC-7) cells were incubated in DMEM medium at 37 °C in a humidified atmosphere containing 5% CO2. Human fibrosarcoma (HT 1080) cells were incubated in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (without Hepes). All of the mediums included 10% FBS and 1% antibiotics (penicillin-streptomycin, 10 000 U/mL). Synthesis and Characterization of Mc-Probe. The Mc-Probe was synthesized manually by standard Fmoc solid phase synthesis method. The amino acids were coupled onto Rink amine resin step by step using HBTU and DIEA as the coupling agent. 20% piperdine/dimethyl formamide (DMF) (v/v) solution was employed to remove Fmoc-protected groups, and 1% TFA/DCM (v/v) was employed to remove Mtt-protected groups. The probe was cleaved from the resin by using 95% TFA/DI water (v/v) for 100 min. After that, the concentrated filtrate obtained was participated in cold ether. Finally, the product was dried under vacuum and saved in -20oC freezer. The probe was purified and examined by HPLC and its molecular weight was confirmed by ESI-MS. Detailed synthesis processes were illustrated in Scheme S1. MMP-2 Responsiveness of Mc-Probe. Mc-Probe (0.5 µM) in PBS was mixed with MMP-2 at the concentration of 10 ng/mL, 20 ng/mL, 40 ng/mL, 100 ng/mL or 200 ng/mL, respectively. As controls, Mc-Probe (0.5 µM) was incubated without MMP-2, or it was incubated with MMP-2 (100 ng/mL) preprocessed by 1,10-phenanthroline monohydrate (50 μM). The catalytic function of MMP-2 was preprocessed by co-culture with its inhibitor (1,10phenanthroline monohydrate, 50 μM) for 1 h at 37 oC. The fluorescence intensity of the mixture was recorded by


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LS55 luminescence spectrometer at predetermined time intervals respectively. Caspase-3 Responsiveness of Mc-Probe. Mc-Probe (0.5 µM) in PBS was mixed with caspase-3 at the concentration of 0.025 U/mL, 0.05 U/mL, 0.1 U/mL, 0.2 U/mL or 0.5 U/mL, respectively. As controls, Mc-Probe (0.5 µM) was incubated without caspase-3, or it was incubated with caspase-3 (0.5 U/mL) preprocessed Ac-DEVD-CHO (50 μM). The catalytic function of caspase-3 was preprocessed by co-culture with its inhibitor (Ac-DEVD-CHO, 50 μM) for 1 h at 37 oC. The fluorescence intensity of the mixture was recorded by LS55 luminescence spectrometer at predetermined time intervals respectively. Besides, caspase-9 responsiveness of Mc-Probe was performed using the same method. Cytotoxicity Assay. The cytotoxicity of Mc-Probe against COS7 cells, HT 1080 cells and SCC-7 cells were measured by MTT assay. Briefly, COS7 cells, HT 1080 cells and SCC-7 cells were cultured on 96-well plates with 6000 cells/well for 24 h in 100 µL culture medium. And then, gradient concentrations of Mc-Probe in culture medium were added into the well, respectively. After 48 h, 20 µl of MTT (5 mg/mL in PBS buffer) was added into each well. 4 h later, the culture medium was replaced with 150 µl DMSO. The optical intensity (OD, at 570 nm) of each well was recorded by Microplate reader (BIO-RAD 550). The cell viabilities were calculated as: cell viability (%) = (OD(samples)/OD(control)) ×100. OD(samples) and OD(control) represented the optical intensities in the presence and absence of Mc-Probe at 570 nm. The cytotoxicity of cisplatin against COS 7 and SCC-7 cells were measured using the same method. Besides, the cytotoxicity of Mc-Probe (5 µM) with or without caspase-3 inhibitor (50 µM) was also measured by MTT assay. Confocal Laser Scanning Microscope (CLSM) Observations. Both of the MMP-2 and caspase-3 imaging abilities of Mc-Probe were observed by CLSM. The MMP2 imaging ability of Mc-Probe was analyzed using COS7 cells, HT 1080 cells and SCC-7 cells. Briefly, COS7 cells, HT 1080 cells and SCC-7 cells were seeded on Petri dishes and incubated for 24 h, respectively. After which, the cells were co-cultured with Mc-Probe (5 µM) for 6 h. And then, the cells were washed trice with PBS and cultured in fresh medium. Finally, the cells were observed by CLSM. Additionally, COS7 cells, HT1080 cells and SCC-7 cells after treatment with Mc-Probe for 6 h in the presence of MMP2 inhibitor were also observed by CLSM as controls. The caspase-3 imaging ability of Mc-Probe was analyzed using COS7 cells and SCC-7 cells. Briefly, after cultured in Petri dishes for 24 h, the cells were treated with Mc-Probe (5 µM) for 6 h. And then, the cells were washed trice with PBS. The cells were incubated with cisplatin (5 µM) or without any treatment as controls. Besides, the cells were treated with caspase-3 inhibitor (Ac-DEVD-CHO, 50 µM) and cisplatin as controls. After 12 h, the cells were observed by CLSM (Green Channel: Ex: 488 nm, Em: 515 ± 35 nm. Red Channel: Ex: 543 nm, Em: 650 ± 35 nm).

Flow Cytometry Analysis. The MMP-2 and caspase-3 imaging abilities of Mc-Probe were also analyzed by flow cytometry. Briefly, COS7 cells, HT 1080 cells and SCC-7 cells were incubated with Mc-Probe (5 µM) and the cells without any treatment were used as controls. After 6 h, the cells were washed trice with PBS. And then, the cells were digested by trypsin, and the collected cells were resuspended in PBS. Finally, the fluorescence of the cells was analyzed by flow cytometry. The caspase-3 imaging ability of Mc-Probe was analyzed by using COS7 cells and SCC-7 cells. Briefly, COS7 cells and SCC-7 cells were incubated with Mc-Probe (5 µM) for 6 h. After washing with PBS, the cells were incubated in cisplatin (5 µM) contained culture medium for 12 h. After which, the cells were washed with PBS and analyzed by flow cytometry. Furthermore, Mc-Probe was also used to analysis the STS induced cell apoptosis by flow cytometry. Briefly, SCC-7 cells were incubated with Mc-Probe (5 µM) for 6 h. And then, the cells were treated with STS (2 µM) for 0 h, 1 h, 2 h, 3 h and 4 h, respectively. After which, the cells were washed by PBS, and the collected cells were analyzed by flow cytometry. The cells without any treatment were used as a control.

Figure 1. Fluorescence emission spectra of Mc-Probe (0.5 µM) after treatment with (A) MMP-2 (200 ng/mL) or (B) caspase-3 (0.5 U/mL). Time dependent fluorescence recovery of Mc-Probe (0.5 µM) after treatment with (C) MMP-2 or (D) caspase-3 at various concentrations in the presence or absence of their corresponding inhibitor. FI0: fluorescence intensity of Mc-Probe at 0 h. FIt: fluorescence intensity of Mc-Probe at various time points. (E)MMP-2 concentration and (F) caspase-3 concentration related fluorescence recovery of Mc-Probe. Excitation wavelength: 465 nm for FAM and 540 nm for TAMRA.



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Emission wavelength: 520 nm for FAM and 570 nm for TAMRA.

RESULTS AND DISCUSSION

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assay. As expected, Mc-Probe exhibited a good biocompatability (Figure S13). And then, the MMP-2 imaging ability was evaluated in COS7, HT1080 and SCC-7 cell lines.

The probe was obtained by solid phase peptide synthesis (SPPS) method (Scheme S1). The structure of McProbe was verified by electrospray ionization-mass spectrometry (ESI-MS) (Figure S1-S2). Furthermore, the purity of Mc-Probe (92.5%) was analyzed by high performance liquid chromatography (Figure S3). As we know, the distance between the donor and the acceptor, and also the spectra overlap of the fluorophores could dramatically influence the efficiency of the FRET process.28 Firstly, the fluorescence response of Mc-Probe against MMP-2 and caspase-3 were evaluated. As illustrated in Figure 1A, a 4.77-fold TAMRA fluorescence recovery was observed after treatment with MMP-2. And also, a 45.99-fold FAM fluorescence recovery was observed after treatment with caspase-3 (Figure 1B). However, no fluorescence changes were observed when without the treatments of MMP-2 (Figure S4) and caspase-3 (Figure S5) or treatment with caspase-9 (Figure S6). These results indicated that Mc-Probe kept stable in the absence of these enzymes and it has fast response abilities and selectivity against MMP-2 and caspase-3 with distinct fluorescence emissions and enhanced fluorescence intensity. Moreover, it was observed that the fluorescence quenching efficiency was higher than the single FRET pair based fluorescent probe,32,33 indicating that multi-FRET approach could extensively improve the signal-to-noise ratio for FRET based fluorescence imaging. This phenomenon was mainly ascribed to the improved absorption efficiency among FAM/TAMRA/Dabcyl fluorophore pairs, which was verified by their UV-vis absorbance (Figure S7). Furthermore, a slightly FAM fluorescence recovery was also observed after treatment with MMP-2 (Figure S8-S9) and negligible TAMRA fluorescence changes was observed after treatment with caspase-3 (Figure S10). These results were reasonable because Dabcyl acted as the acceptor for both TAMRA and FAM, which further demonstrated the multi-FRET processes in Mc-Probe. Besides, Mc-Probe exhibited a concentration related fluorescence recovery against MMP-2 (Figure 1C) and caspase-3 (Figure 1D), and negligible fluorescence recovery was observed after treatment with enzymes in the presence of their corresponding inhibitors. And the fluorescence recovery of Mc-Probe exhibited the positive linear relation with MMP-2 concentration (Figure 1E), caspase-3 concentration (Figure 1F and Figure S11) and incubation time (Figure S12). These results indicated the specificity and the potential of Mc-Probe for quantitative MMP-2 and caspase-3 imaging. The expression of MMP-2 was varies in normal cell (COS7) and cancer cell (HT1080 and SCC-7) lines.34,35 Before investigating the MMP-2 imaging ability of Mc-Probe in living cells, its cytotoxicity was conducted using MTT

Figure 2. CLSM images of COS7 cells, HT1080 cells and SCC-7 cells after treatment with Mc-Probe (5 µM) for 6 h in the (A-C) absence and (D-F) presence of MMP-2 inhibitor. Scale bar: 20 µm. (G) Western blot and (H) the quantative analysis of MMP-2 expression in SCC-7, COS7 and HT1080 cell lines. (I) Flow cytometry analysis of the TAMRA fluorescence in COS7, SCC-7 and HT1080 cells after treatment with Mc-Probe (5 µM) for 6 h or without any treatment.


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As shown in Figure 2B1-2C1, intense TAMRA fluorescence was observed in HT1080 cells and SCC-7 cells, and reduced TAMRA fluorescence was observed in COS7 cells (Figure 2A1). However, negligible TAMRA fluorescence was observed in all of the cells after treatment with McProbe in the presence of MMP-2 inhibitor (Figure 2D-F). Similar results were also observed by their quantitative flow cytometry analysis (Figure 2I), indicating that McProbe was suitable for MMP-2 related cancer cell diagnosis and imaging. Furthermore, negligible FAM fluorescence was observed in all of the cells (Figure 2A2-2F2 and Figure S14). These results confirmed that the MMP-2 imaging process was independent to caspase-3 imaging process. Moreover, the MMP-2 expression in COS7, HT1080 and SCC-7 cells lines were also analyzed by western blot (Figure 2G). In accordance with our confocal laser scanning microscope (CLSM) and flow cytometry observations, both HT1080 and SCC-7 cell lines had a relative higher level of MMP-2 expression than COS7 cell lines. Furthermore, there was a positive correlation between the expression level of MMP-2 and the TAMRA fluorescence recovery observed in SCC-7, COS7 and HT1080 cell lines (Figure 2H), strongly demonstrating that Mc-Probe could be used for MMP-2 imaging in living organisms. The overexpression of caspase-3 was closely associated with some pathological diseases, and most of the therapeutics (chemotherapy, photothermal therapy, photodynamic therapy, et al.) could induce cell death by caspase-3 related apoptosis pathway.12,36,37 Thus, caspase-3 imaging could not only used for disease diagnosis, but also for the early therapeutic efficacy evaluations.38 Firstly, the caspase-3 imaging ability of Mc-Probe was evaluated against SCC-7 cells by flow cytometry using staurosporine (STS) as a apoptosis inducer. As illustrated in Figure S15, enhanced FAM fluorescence recovery was observed when prolonging the incubation time of STS, which indicated that Mc-Probe could indeed be used for caspase-3 imaging. The different degrees of FAM fluorescence recovery were ascribed to the various proteolysis degrees of McProbe, which was determined by the levels of caspase-3 and the incubation time (Figure 1D, 1F). As one of the mostly used anti-cancer drugs, cisplatin could trigger cell death by apoptosis mechanism (Figure S16-S17). To further investigate the spatiotemporal MMP-2 and caspase-3 imaging ability of Mc-Probe, the fluorescence recoveries were observed in MMP-2 negative COS7 cells and MMP-2 positive SCC-7 cells using cisplatin as cell apoptosis inducer. As shown in Figure 3A1-3F1, weak TAMRA fluorescence was observed in COS7 cells and intense TAMRA fluorescence was observed in SCC-7 cells, which were in coincidence with the previous results (Figure 2A1 and 2C1). However, compared with the negligible FAM fluorescence recovery when without the treatment of apoptosis inducer (Figure 3A2 and 3D2), a dramatically FAM fluorescence enhancement was observed after treatment with cisplatin in both COS7 cells (Figure 3C2) and SCC-7 cells (Figure 3F2). The distinct fluorescence recoveries further indicat-

ed that the MMP-2 and caspase-3 imaging ability of McProbe was independent of each other, which could

Figure 3. CLSM images of (A-C) COS7 cells and (D-F) SCC-7 cells after different treatments: (A, D) Mc-Probe (5 µM) for 6 h; (B, E) Mc-Probe (5 µM) for 6 h and cisplatin (5 µM) for another 12 h in the presence of caspase-3 inhibitor; (C, F) Mc-Probe (5 µM) for 6 h and cisplatin (5 µM) for another 12 h in the absence of the caspase-3 inhibitor. Scale bar: 20 µm. Flow cytometry analysis of the FAM fluorescence in (G, I) COS7 cells and (H, J) SCC-7 cells after treatment with Mc-Probe (5 µM) for 6 h and further treatment with (yellow line) or without (cyan line) cispla-



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tin (5 µM) for 12 h, and the cells without any treatment were used as blank control (red line). improve the precision for MMP-2 and caspase-3 related disease diagnosis and cancer theranostics. Besides, no obvious cytotoxicity was found on COS7 and SCC-7 cells after treatment with Mc-Probe in the presence or absence of caspase-3 inhibitor (Figure S18). And the FAM fluorescence kept at a background level after treatment with cisplatin in the presence of caspase-3 inhibitor (Figure 3B2 and 3E2, Figure S19), further confirming that the FAM fluorescence recovery was indeed ascribed to the proteolysis of Mc-Probe by caspase-3 (Figure 1D). Moreover, similar results were also obtained by flow cytometry when with or without the treatment of cisplatin in COS7 cells and SCC-7 cells (Figure 3G-3J, Figure S20). These results indicated that Mc-Probe could be used as a robust fluorescent probe for spatiotemporal MMP-2 and caspase-3 imaging with distinct fluorescence emissions, which could pave the way for precise disease diagnosis and apoptosis related drug screening.

SCC-7 cells; Z-stack images of COS7 cells after treatment with Mc-Probe; Flow cytometry analysis of the TAMRA fluorescence in COS7 cells and SCC-7 cells after treatment with Mc-Probe and cisplatin; This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51690152 and 51233003) and the Ministry of Science and Technology of China (2016YFC1100703).

REFERENCES 1.

Conclusions

2.

In conclusion, we designed and characterized a novel fluorescent probe (Mc-Probe) for simultaneous MMP-2 and caspase-3 imaging. Owing to the multi-FRET processes and the specificity of the peptide linker, Mc-Probe could simultaneously image MMP-2 and caspase-3 activities with high selectivity and signal-to-noise ratios. Furthermore, the overexpression of MMP-2 in cancer cell lines and the cisplatin induced cell apoptosis was spatiotemporal imaged with distinct fluorescence emissions. Because of the independent process of Mc-Probe for MMP-2 and caspase-3 imaging, Mc-Probe could meet the demands for precise disease diagnosis and cancer theranostic applications, which would be a versatile platform for multi-complex biological imaging.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

ASSOCIATED CONTENT Supporting Information. Synthetic details; ESI-MS and HPLC of Mc-Probe; Fluorescence emission spectra of McProbe without the treatment of MMP-2 and caspase-3; Fluorescence emission spectra of FAM in the probe after treatment with caspase-9; Fluorescence emission spectrum of FAM and the UV-vis absorbance of Dabcyl, TAMRA; Time related TAMRA fluorescence recovery of Mc-Probe after treatment with caspase-3; Fluorescence emission spectra of FAM in Mc-Probe after treatment with MMP-2; Fluorescence emission spectra of TAMRA in Mc-Probe after treatment with caspase-3; Time related and caspase-3 concentration related FAM fluorescence recovery of Mc-Probe; The cytotoxicity of Mc-Probe in HT1080, SCC-7 and COS7 cells; Z-stack images of SCC-7 cells after treatment with the Mc-Probe; Flow cytometry analysis of the FAM fluorescence of SCC-7 cells after treatment with Mc-Probe and STS; The cytotoxicity of cisplatin in COS7 cells and SCC-7 cells; Western blot of caspase-3 expression in COS7 and SCC-7 cells after treatment with cisplatin; The cytotoxicity of Mc-Probe (5 μM) or caspase-3 inhibitor (Ac-DEVD-CHO, 50μM) in COS7 cells and

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8 ACS Paragon Plus Environment

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