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Intracellular Disassembly of Self-Quenched Nanoparticles Turns NIR Fluorescence on for Sensing Furin Activity in Cells and in Tumors Yue Yuan, Jia Zhang, Qinjingwen Cao, Linna An, and Gaolin Liang* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China S Supporting Information *

ABSTRACT: There has been no report on enzyme-controlled disassembly of self-quenched NIR fluorescent nanoparticles turning fluorescence on for specific detection/imaging of the enzyme’s activity in vitro and in vivo. Herein, we reported the rational design of new NIR probe 1 whose fluorescence signal was self-quenched upon reduction-controlled condensation and subsequent assembly of its nanoparticles (i.e., 1-NPs). Then disassembly of 1-NPs by furin turned the fluorescence on. Employing this enzymatic strategy, we successfully applied 1-NPs for NIR detection of furin in vitro and NIR imaging furin activity in living cells. Moreover, we also applied 1-NPs for discriminative NIR imaging of MDA-MB-468 tumors in nude mice. This NIR probe 1 might be further developed for tumor-targeted imaging in routine preclinical studies or even in patients in the future. their fluorescence while disassembly of the dyes recovers the fluorescence again.20,21 Nevertheless, NIR off−on switch probes based on the disassembly strategy were very rarely reported,22 and to the best of our knowledge, there is no enzyme-controlled disassembly of NIR probe reported. The trans-Golgi protease furin is one kind of protein convertase playing important roles in homeostasis and in diseases ranging from Alzheimer’s disease to anthrax, Ebola fever, and cancer.23 Several cancers, including nonsmall-cell lung carcinomas, squamous-cell carcinomas of the head and neck, and glioblastomas, are reported to overexpress furin.24 Furin preferentially cleaves Arg-X-Lys/Arg-Arg↓X peptide substrates (X can be any amino acid residue and ↓ indicates the cleavage site), which benefits chemists to design different substrates for its cleavage.25 So far, many probes, based on this specific substrate, for furin activity imaging (including optical imaging,26,27 photoacoustic imaging,28 magnetic resonance imaging,29 and nuclear imaging30) have been developed. On the basis of the literature research above, as shown in Figure 1, we rationally designed a new NIR compound 1 whose fluorescence is self-quenched upon reduction-controlled condensation and self-assembly of its nanoparticles (i.e., 1NPs). When 1-NPs were subjected to furin cleavage and disassembly, the fluorescence was recovered, with which we successfully applied 1-NPs for NIR imaging furin activity in MDA-MB-468 cancer cells. Moreover, we also intravenously injected 1-NPs to nude mice xenografted with MDA-MB-468

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ear-infrared (NIR)-fluorescence probes have shown great potential for in vivo imaging in recent years due to the high penetration ability of their excitations (or emissions) through tissues in the NIR range (650−900 nm), while in the meantime maximally avoiding biological autofluorescence background.1−4 Compared to inorganic NIR probes, organic NIR fluorescent dyes are emerging as very attractive candidates for in vivo imaging due to their improved photophysical properties and availability for large-scale chemical synthesis.5,6 Moreover, it is not difficult to modify these organic dyes with functional groups to conjugate biomolecules such as DNA, peptides, proteins, or antibodies for molecular imaging their corresponding biomarkers in vitro, in cells, or in vivo.7−9 Specifically, once the NIR dyes are covalently or noncovalently conjugated with cancer-targeting warheads, they could be applied promisingly to imaging cancers with high specificity and sensitivity. To date, fluorescence always-on probes have been used for most of the imaging studies. But as we known, unspecific endocytosis of fluorescence always-on probes in nontargeted cells leads to a false positive signal.10 Compared to always-on signals, an “on−off” or “off−on” switch signal should greatly help to address the above issue and be more suitable for celltargeted detection due to its enhanced optical spatial resolution.11−13 On the basis of the fluorescence changes, several “off−on” or “on−off” switches have been successfully developed for the detections of anions, metal ions, nucleotide, proteins, etc.14−19 Between these two types of switches, off−on is advantageous over on−off in fluorescence imaging owning to its lower background signal. Assembly and disassembly are two most prevalent and important processes in cells and recently have been employed to develop fluorescent switches since assembly of fluorescence dyes always cause self-quenching of © XXXX American Chemical Society

Received: February 28, 2015 Accepted: May 19, 2015

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Figure 1. Chemical structures of furin-activable 1 and dye FPR-675.

(CBT) was synthesized following the literature method.31 The synthetic route for 1 is shown in Scheme S1 of the Supporting Information). Synthesis of B: Compound A was synthesized with solid phase peptide synthesis (SPPS). Isobutyl chloroformate (IBCF, 38.0 mg, 0.278 mmol) was added to a mixture of compound A (397 mg, 0.200 mmol) and 4-methylmorpholine (MMP, 52.0 mg, 0.514 mmol) in THF (5.00 mL) at 0 °C under N2. The reaction mixture was stirred for 40.0 min. The solution of 2cyano-6-aminobenzothiazole (CBT, 40.0 mg, 0.229 mmol) was added to the reaction mixture and further stirred at 0 °C for 1 h. Then the mixture was stirred overnight at room temperature. Compound B (265 mg, yield: 61.9%) was purified with HPLC using water−acetonitrile added with 0.1% TFA as the eluent (from 15:85 to 0:100). MS: calculated for B [(M + 2H)2+], 2141.91; obsd ESI-MS, m/z 1071.42, z = 2. Synthesis of C: The Boc and Pbf protecting groups of compound B were removed with dichloromethane (DCM, 1 mL) and triisopropylsilane (TIPS, 200 μL) in TFA (19 mL) for 3 h. Compound C (144 mg, yield: 90.6%) was obtained after HPLC purification using water−acetonitrile added with 0.1% TFA as the eluent (from 5:5 to 5:95). MS: calculated for C [(M + H)+], 1284.61; obsd ESI-MS, m/z 1284.70. Synthesis of D: FPR-675 (10.6 mg, 0.01 mmol) were welldispersed in 2 mL DMF, then compound C (25.7 mg, 0.020 mmol) and N,N-Diisopropylethylamine (DIPEA, 2.6 mg, 0.02 mmol) were added into the mixture and further stirred for 6 h at RT to yield compound D. Compound D (19.4 mg, yield: 82.7%) was purified with HPLC using water−acetonitrile added with 0.1% TFA as the eluent (from 5:5 to 5:95). MS: calculated for D [(M + H)+], 2345.83; obsd MALDI-TOF/MS, m/z 2345.84. Synthesis of 1: The Fmoc protecting group of compound D was cleaved with 5% piperidine in DMF (4 mL) at 0 °C for 5 min, then 200 μL TFA was added to neutralize the alkaline, thus compound 1 (14.9 mg, yield: 84.9%) was obtained after HPLC purification with water−acetonitrile as the eluent (from 99:1 to 35:65) and sent for high-resolution (HR) mass spectrum analysis. MS: calcd for C91H140N22O21S8 [(M + H)+],

tumors and LoVo tumors for discriminative NIR imaging of MDA-MB-468 tumors in vivo.

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EXPERIMENTAL SECTION Materials. All the starting materials were obtained from Sigma or Adamas. Commercially available reagents were used without further purification, unless noted otherwise. All chemicals were reagent grade or better. FPR-675 was obtained from BioActs (Korea). Furin was purchased from Biolabs [one unit (U) corresponds to the amount of furin that releases 1 pmol of methylcoumarinamide (MCA) from the fluorogenic peptide Boc-RVRR-MCA (Bachem) in 1 min at 30 °C]. General Methods. Matrix-assisted laser desorption (MALDI) ionization-time-of-flight (TOF)/TOF and ESI mass spectra were obtained on a time-of-flight Ultrflex II mass spectrometer (Bruker Daltonics) and on a Finnigan LCQ Advantage ion trap mass spectrometer (ThermoFisher Corporation) equipped with a standard ESI source, respectively. High performance liquid chromatography (HPLC) purification was performed on a Shimazu UFLC system equipped with two LC-20AP pumps and an SPD-20A UV/vis detector using a Shimazu PRC-ODS column. HPLC analyses were performed on an Agilent 1200 system equipped with a G1322A pump and in-line diode array UV detector an Agilent Zorbax 300 SD-C18 RP column, with CH3CN (0.1% of TFA) and water (0.1% of TFA) as the eluent. Cell images were obtained on the IX71 fluorescence microscope (Olympus, Japan). Transmission electron micrograph (TEM) images were obtained on a JEM-2100F field emission transmission electron microscope operated at an acceleration voltage of 200 kV. Fluorescence spectra were recorded on a F-4600 fluorescence spectrophotometer (Hitachi High-Techonologies Corporation, Japan) with excitation wavelength set to 665 nm. Cells were routinely cultured in Dul-becco’s modified Eagle’s medium (DMEM, Hycolon) supplemented with 10% fetal bovine serum at 37 °C, 5% CO2, and humid atmosphere. 4−6 Week old (weighting 19−20 g) BALB/c nude mice were used for animal experiments. Synthesis and characterization of 1 (Cys(StBu)-Arg-Val-ArgArg-Lys(FPR-675)-CBT). 2-Cyano-6-aminobenzothiazole B

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recoveries of the fluorescence, reaching to 53.3% of its original of 1 (Figure 2A). When the amount of furin was increased from 10 to 25 pmol U1−, the incubation time could be shortened from 5 h to 2 h to achieve similar fluorescence recovery (as shown in Figure S2 of the Supporting Information). Transmission electron microscope (TEM) observations were performed to characterize 1-NPs formed with 1 treated by TCEP, and their subsequent incubation with furin. As shown in Figure 2B, after 1 h incubation of 1 in the presence of TCEP at 37 °C, as-formed nanoparticles (i.e., 1-NPs) have an average diameter of 20.5 ± 9.1 nm (size ± SD, 87 nanoparticles analyzed) (Figure S3 of the Supporting Information). After 5 h incubation of 1-NPs with furin, TEM image indicated that most of nanoparticles were digested by furin and disassembled (Figure 2C), while the morphology of the leftover nanoparticles became irregular and smaller with an average diameter of 16.3 ± 13.5 nm (size ± SD, 30 nanoparticles analyzed (Figure S4 of the Supporting Information). The above selfassembly and disassembly of 1-NPs were also confirmed with dynamic light scattering (DLS) analysis (Figure S5 of the Supporting Information). To chemically validate the TCEPcontrolled condensation and subsequent furin cleavage, we directly injected the above two incubation mixtures into a highperformance liquid chromatography (HPLC) system and collected the peaks for matrix-assisted laser desorption/ ionization (MALDI) mass spectroscopic analyses. As shown in Figure 2D, HPLC trace of the reaction mixture of 12 μM 1 that incubated with 3 mM TCEP at 37 °C for 1 h only show one main peak with retention time of 22.8 min, identified as the condensation product of 1 (i.e., 1-Dimer, Figure S6 of the Supporting Information), indicating that 1-NPs were composed of 1-Dimer. After 5 h incubation of 1-NPs with furin (10 pmol U1−) at 37 °C, we found that 69.7% of 1-Dimer in 1-NPs was cleaved by furin to yield 1-D-Cleaved (Figure S7 of the Supporting Information) with a retention time of 22.2 min on the HPLC trace, while 24.1% of 1-Dimer was unacted (Figure 2D), echoing very well with the TEM observation in Figure 2C. Kinetic study indicated that the Kcat and Kcat/KM values of furin toward D were 8.0 folds and 4.7 folds of those toward 1-NPs, respectively (Figure S8 of the Supporting Information). This suggests that 1-NPs are harder for furin cleavage than the small molecular precursor. To test the stability of 1-NPs in cells and in mice, we incubated 1-NPs with DMEM for 24 h or fetal bovine serum (FBS) for 72 h at 37 °C. HPLC traces indicated that 1-NPs were very stable during the time windows for incubation (Figure S9 of the Supporting Information). NIR Imaging of Furin-Like Activity in Cells. To demonstrate the efficacy of 1-NPs for imaging furin activity, we first tested them on living cells. Furin-overexpressing breast adenocarcinoma MDA-MB-468 cells and furin-deficient colorectal adenocarcinoma LoVo cells (proven by Western blotting, Figure S10 of the Supporting Information), were chosen for the following experiments. High expression of furin in MDA-MB468 cells was also confirmed with immunofluorescence staining of furin using Alexa Fluor 488-labeled secondary antibody. An overlay of the fluorescence staining of furin (green) with 4′,6diamidino-2-phenylindole (DAPI) staining of nucleus (blue) clearly shows the locations of furin (i.e., the Golgi bodies) as reported (Figure 3).23 Before imaging experiments, MDA-MB468 cells or LoVo cells were starved for 4 h and then cultured in 2 mL of normal culture medium containing 100 μL 1-NPs dispersion or 100 μL FPR-675 (10 μM) for 12 h, respectively. Fluorescence microscopic imaging of MDA-MB-468 cells

2123.76248; obsd HR-MALDI-TOF/MS, m/z 2123.76248 (Figure S1 of the Supporting Information).

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RESULTS AND DISCUSSION Rationale of the Design. We designed our probe with the following components, as shown in Figure 1: a disulfided cysteine (Cys) motif, a 2-cyanobenzothiazole (CBT) motif, a RRVR substrate for furin cleavage, and a FPR-675 motif conjugating to the side chain of a lysine (Lys) motif to generate the NIR signal. In the presence of reducing agents, disulfide bond of compound 1 will be reduced to expose 1,2-aminothiol group, which instantly condenses with the cyano group of the CBT motif to yield macrocyclized oligomers (mostly dimers). Subsequently, the oligomers will self-assemble into nanoparticles (1-NPs), as we demonstrated previously.32−34 Thus, the fluorescence of 1 from FPR-675 motif is self-quenched. However, the existence of furin will disassemble 1-NPs into a new monomer (i.e., 1-D-Cleaved), recovering 53.3% of its original fluorescence intensity (FL). The turn-on FL from 1NPs was successfully applied for NIR detection of furin activity in vitro and in cells and discriminative NIR imaging MDA-MB468 tumors in vivo. In Vitro NIR Fluorescence Detection of Furin Activity with 1-NPs. After synthesis of 1, we used tris(2-carboxyethyl)phosphine (TCEP) to trigger its condensation to assemble 1NPs and applied as-formed 1-NPs for NIR fluorescence detection of furin in vitro. In detail, 12 μM 1 in the pH 7.4 buffer containing 37.5% DMSO (v/v) was incubated in the presence of 3 mM TCEP at 37 °C for 1 h. After that, the reaction mixtures were centrifuged, and 1-NPs obtained by centrifugation were redispersed in same volume of the furin working buffer containing 10% DMSO for fluorescence measurements. Comparing to the fluorescence spectrum of 1, we noticed that of 1-NPs dropped significantly (4.5-fold) (Figure 2A). Then the 1-NPs dispersion was incubated with furin (10 pmol U1−) at 37 °C for 5 h. We observed obvious

Figure 2. (A) Fluorescence spectra of 100 μL 1 at 12 μM, 100 μL 1NPs dispersion, and 100 μL 1-NPs dispersion treated with furin (10 pmol U1−) in furin working buffer, respectively. Excitation: 665 nm. (B) TEM image of 1-NPs dispersion. (C) TEM image of 1-NPs dispersion after being incubated with 10 pmol U1− Furin at 37 °C for 5 h. (D) HPLC traces of 12 μM 1 (black) in furin working buffer, 100 μL 1-NPs dispersion (red), and the incubation mixture of 100 μL 1NPs dispersion incubated with 10 pmol U1− furin at 37 °C for 5 h (blue). Absorbance: 675 nm. C

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Figure 3. Topmost row, fluorescence, and overlay images of MDA-MB-468 cells after incubation with 100 μL 1-NPs dispersion in 2 mL of culture medium at 37 °C for 12 h. Top middle row, fluorescence and overlay images of LoVo cells after incubation with 100 μL 1-NPs dispersion in 2 mL of culture medium at 37 °C for 12 h. Bottom middle row, fluorescence and overlay images of MDA-MB-468 cells after incubation with 100 μL FPR675 (10 μM) in 2 mL of culture medium at 37 °C for 12 h. Bottommost row, fluorescence and overlay images of LoVo cells after incubation with 100 μL FPR-675 (10 μM) in 2 mL of culture medium at 37 °C for 12 h. Blue is DAPI staining of nucleus, green is immunofluorescence staining of furin, red is from 1-NPs or FPR-675. Scare bar: 10 μm.

Figure 4. In vivo active furin-overexpression-tumor-targeted imaging with 1-NPs. Spectrally unmixed in vivo fluorescence images of tumor-bearing nude mouse at 6, 24, 48, and 72 h after injection of 100 μL 1-NPs. LoVo tumor and MDA-MB-468 tumors are indicated with “L” and “M” by white arrows, respectively. Tumors are in the blue circles.

incubated with 1-NPs clearly shows red roundish fluorescence emissions which well overlap with the green fluorescence of furin staining (topmost row in Figure 3), suggesting that 1-NPs were digested and disassembled by furin inside the cells, turning on the fluorescence on the sites of furin (i.e., the Golgi bodies). For furin-deficient LoVo cells that incubated with 1-NPs, both the green fluorescence from furin staining and the red fluorescence from 1-NPs could be hardly observed (top middle row in Figure 3), suggesting 1-NPs are only susceptible to furin. When these two cell lines were subjected to FPR-675 imaging, red homogeneous fluorescence emissions from MDAMB-468 cells (bottom middle row of Figure 3), which were similar to those from LoVo cells (bottommost row in Figure 3) could be observed, further confirming that the red fluorescence emissions in the above 1-NPs-treated MDA-MB-468 cells

actually resulted from furin-cleaved products of 1-NPs (i.e., 1D-Cleaved) but not from the free FPR-675 yielded from the hydrolysis of 1-NPs before furin cleavage. The zoom out cell images of Figure 3 were shown in Figure S11 of the Supporting Information. Interestingly, when the volume of 1-NPs dispersion for incubation was increased from 100 to 300 μL, the incubation time for cell imaging could be shortened from 12 to 3 h, and their imaging effects were similar (data not shown). Cytotoxicity of 1-NPs on MDA-MB-468 cells and LoVo cells was assessed, and the results indicated that up to 200 μL 1-NPs in 2 mL culture medium did not induce obvious cytotoxicity on the cells for 24 h (Figure S12 of the Supporting Information). NIR Imaging of Furin-Like Activity in Tumor-Bearing Mice. Each nude mouse was subcutaneously implanted with D

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Analytical Chemistry LoVo tumor in the left thigh and MDA-MB-468 tumor in the right thigh. Until the tumor sizes were within 5−10 mm in diameter, the nude mice were randomly divided into 2 groups (n = 3 for each group). 100 μL of 1-NPs (20.2 μg, ten times concentrated) in the furin working buffer, or 100 μL of FPR675 (15 nmol) in phosphate-buffered saline (PBS) at pH 7.4 were respectively injected into each of these tumor-bearing nude mice in each group through tail veins, and the mice were imaged for 72 h in a small animal imaging system. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. As shown in Figure 4, from 6 to 72 h post injection, fluorescent signals from organs in mice injected with 1-NPs gradually decreased, while the fluorescence signals from these two types of tumor gradually increased. At 6 h and 24 h, strong NIR fluorescence was observed from the brain, spleen, and intestine of the mouse. At 72 h, strong NIR-fluorescence emissions with good contrast were observed from the MDA-MB-468 tumors in the right thighs of the nude mice. In contrast, the LoVo tumors in the left thighs only showed very weak NIR-emissions which were comparable to those from other organs or tissues. Quantitative analysis of the fluorescence intensity from MDAMB-468 tumors or LoVo tumors in Figure 4 were provided in Figure S13 of the Supporting Information. For those tumorbearing nude mice injected with FPR-675, neither the MDAMB-468 tumors nor the LoVo tumors showed stronger NIRfluorescence emissions than those from other organs, as shown in Figure S14 of the Supporting Information). These results clearly indicated that our 1-NPs could be effectively uptaken by tumors via enhanced permeability (EPR) effect, and their NIR fluorescence could be selectively turned on by furin-overexpressing tumors, offering discriminative tumor-targeted NIR imaging in vivo. To further validate that strong NIR-fluorescence in 1-NPsteated mice was actually emitted from MDA-MB-468 tumors, after imaging of these mice at 72 h post injection, we sacrificed the mice and took out the tumors and organs from their bodies and conducted ex vivo imaging. As shown in Figure 5, the MDA-MB-468 tumors showed highest NIR-fluorescence among the organs studied, while the LoVo tumors only exhibited weak NIR-fluorescence which was comparable to that of kidneys or spleens. The lungs showed the lowest fluorescence. In contrast, neither MDA-MD-468 tumors nor

LoVo tumors in FPR-675-treated mice showed distinguishable fluorescence from other organs (Figure S15 of the Supporting Information), consistent with the whole body imaging observations of the mice (Figure S14 of the Supporting Information).

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CONCLUSION In summary, by the rational design of a biocompatible compound 1, we have developed a “smart” method of furincontrolled disassembly of its nanoparticles (i.e., 1-NPs) that turns the fluorescence signals “on” for selective detection of furin activity in vitro and in cells. With the good contrast of fluorescent signal obtained in cells, we also successfully used 1NPs for discriminative imaging of furin activity in mice. We envision that the in vivo imaging time in this work (i.e., 72 h) could be greatly shortened, taken our NIR probe 1-NPs be modified with some tumor-targeting warheads (e.g., RGD, folic acid, antibody, etc.). These in vitro and in vivo studies suggest that the NIR probe 1-NPs might be developed for tumortargeted imaging in routine preclinical studies or even in patients.

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ASSOCIATED CONTENT

S Supporting Information *

HR-MALDI-TOF/MS spectrum of 1; statistical results of diameters of nanoparticles in TEM images; dynamic light scattering (DLS) analysis of 1-NPs; HR-MALDI-TOF/MS spectrum of 1-Dimer; HR-MALDI-TOF/MS spectrum of 1-DCleaved; kinetic assay of furin; stability and cytotoxicity studies of 1-NPs; Western blot results; in vivo active furin-overexpression-tumor-targeted imaging with FPR-675; ex vivo fluorescence images of different organs from tumor-bearing nude mice after being intravenously injected with FPR-675; HPLC conditions for the purification of 1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01656.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+86)-551-63600730. Author Contributions

Y.Y. performed syntheses, characterizations, cell imaging, and animal imaging. J.Z. helped with nanocharacterizations and animal imaging. Q.C. helped with the syntheses of the probes. L.A. helped with cell imaging. G.L. designed this project and wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology, Science Research Grant of Hefei Science Center of CAS (2015SRG-HSG037), and National Natural Science Foundation of China (Grants 21175122 and 21375121).

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Figure 5. Ex vivo fluorescence images of different organs from tumorbearing nude mice after being intravenously injected with 100 μL 1NPs for 72 h and imaged (in Figure 4).

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DOI: 10.1021/acs.analchem.5b01656 Anal. Chem. XXXX, XXX, XXX−XXX