Targeted Delivery of a Î³-Glutamyl Transpeptidase ... - ACS Publications

Subscriber access provided by READING UNIV

Article

Targeted Delivery of #-Glutamyl Transpeptidase-Activatable Near-Infrared Fluorescence Probe for Selective Cancer Imaging Zhiliang Luo, Zheng Huang, Ke Li, Yidan Sun, Jianguo Lin, Deju Ye, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05022 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Targeted Delivery of γ-Glutamyl Transpeptidase-Activatable NearInfrared Fluorescence Probe for Selective Cancer Imaging Zhiliang Luo,† Zheng Huang,† Ke Li,‡ Yidan Sun,† Jianguo Lin,*,‡ Deju Ye,*,† and Hong-Yuan Chen† †

State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, Chin. Tel/Fax: +86-25-89681905; E-mail: [email protected] ‡

Key Laboratory of Nuclear Medicine of Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China. Tel/Fax: +86-510-85514422; E-mail: [email protected].

ABSTRACT: Non-invasive and specific detection of cancer cells in living subjects have been essential for the success of cancer diagnosis and treatment. Herein, we reported a strategy of combining a αvβ3 integrin receptor-targetable ligand c-RGD with γglutamyl transpeptidase (GGT)-recognitive substrate γ-glutamate (γ-Glu) to develop a tumor targeting and GGT-activatable nearinfrared (NIR) fluorescence probe for non-invasive imaging of tumors in living mice. We demonstrated that the probe was fluorescence off initially, but the γ-Glu in the probe was specifically cleaved by GGT, releasing fluorescent product that could be selectively taken up by U87MG tumor cells via the αvβ3 receptor-mediated endocytosis. Remarkably enhanced intracellular NIR fluorescence distributed mainly the lysosomes was observed in the tumor cells only, capable of differentiating the tumor cells from GGTpositive but αvβ3-deficient normal cells. Moreover, the probe also showed a high selectivity for the real-time and non-invasive detection of GGT activity in the xenograft U87MG tumors following i.v. administration. This study reveals the advantage of using a combination of receptor-mediated cell uptake with molecular target-triggered activation to design molecular probes for improved cancer imaging, which could facilitate the effective cancer diagnosis.

some cancer cells, were developed.20 Although enhanced cancer imaging was achieved, these dual-targeting imaging probes can also enter into certain normal cells with high expression of either type of receptor. Alternately, the combination of active imaging probe with activatable imaging probe to design new dual-targeting imaging probes, which consist of a receptorrecognitive ligand and a molecular target-responsive substrate conjugated to an “off” imaging tag, have been proposed.21-27 These probes take the advantages of receptor-mediated uptake and molecular target-triggered activation of imaging signals, enabling high specificity and sensitivity for cancer imaging. One intriguing example of using a tumor-targeting ligand (e.g., folic acid) itself as a quencher that was conjugated to a NIR fluorophore via a molecular target (e.g., cathepsin B) cleavable linker to design an activatable fluorescence probe has recently been reported by Choi and coworkers, demonstrating the great promise for selective imaging of ovarian cancers.28-30 γ-Glutamyltranspeptidase (GGT) is an important peptidase participating in the cellular homeostasis of glutathione (GSH) and cysteine (Cys), which is closely related to many essential physiological processes. It is also recognized that the activity of GGT is significantly upregulated in many different diseases, including diabetes,31 asthma,32 and cancer,33-35 and the aberrant expression of GGT can represent as a potential cancer biomarker. The accurate detection of GGT activity is highly desirable for the early diagnosis and predicting treatment efficacy. Accordingly, there have been many activatable imaging probes developed for the detection of GGT activity, most of

INTRODUCTION In the past few years, a number of molecular imaging probes capable of targeting to different biomarkers associated to cancer cells have been developed, offering important tools for the early detection of cancers and the rapid monitoring of therapy response.1-6 These imaging probes can be commonly classified into two main types based on the imaging mechanism. The first one is called active imaging probe, in which an “always on” imaging tag is conjugated to a ligand that can bind to a receptor overexpressed on the cancer cell surface. The imaging mechanism depends on the selective accumulation and retention in the cancer cells via receptor-mediated active delivery and cellular uptake of the probes.7-11 The second one is called activatable imaging probe, where an “off” imaging tag is conjugated to a substrate that is responsive to a molecular target in cancer cells. The imaging mechanism relies on the specific switching “on” imaging signals in response to an effective molecular trigger in the cancer cells.12-16 Both of these two types of imaging probes have found successful applications for cancers imaging, however, they may still suffer from low specificity due to the cross-reactivity with certain normal cells (non-tumorous cells) that share similar characteristics of cancer cells.17 To overcome this limitation, molecular imaging probes using a combination of two different targeting ligands have been proposed to trigger enhanced uptake into cancer cells.18,19 For example, luminescent gold nanoclusters, which are conjugated with both cyclic RGD (c-RGD) and aptamer AS1411 capable of targeting to respective αvβ3 integrin and nucleolin receptors overexpressed on the surface of 1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

which are designed by simply linking a GGT-cleavable substrate (γ-Glu) with a quenched fluorophore.36-41 Upon GGTtriggered removal of γ-Glu in the probes, the fluorescence can be switched “on” from an “off” state, allowing to report on the GGT activity in real time. Very recently, this mechanism has also been employed by us to develop a stable, but GGTactivatable near-infrared fluorescence probe (GANP),42 which showed a high capacity of non-invasive imaging of GGT activity in different tumor cells and in living mice following direct injection into xenograft tumors. Unfortunately, the further application of GANP for extensive in vivo imaging was limited by the poor solubility under physiological conditions and insufficient delivery into tumor tissues. Moreover, GANP and other reported GGT-activatable probes also suffered from cross-reactivity with some normal organ tissues, such as kidney, liver and intestines that have high GGT activity as well.43,44 As such, there is still lack of GGT-activatable imag-

ing probes capable of intravenous (i.v.) injection and selective delivery into tumors for non-invasive and specific imaging of tumors in vivo. Herein, we reported a new tumor-targeting and GGTactivatable NIR fluorescence probe (1) for the specific detection of GGT activity in the tumor of living mice by incorporating the αvβ3 integrin receptor targeting ligand c-RGD and the GGT-cleavable substrate γ-Glu into a pre-quenched NIR fluorophore (Figure 1a). We demonstrated that probe 1 was water soluble and fluorescence off, but could be efficiently activated by GGT to form highly fluorescent probe 2, which could be selectively taken up by tumor cells via the αvβ3 integrin receptor-mediated endocytosis. Therefore, remarkably enhanced intracellular NIR fluorescence was observed in tumor cells only, providing improved selectivity for noninvasive tumor imaging in vivo following i.v. administration.

Figure 1. General design of the dual-targeting NIR fluorogenic probe 1. (a) Chemical structure and proposed GGT-triggered conversion of probe 1 to fluorescent product 2. (b) Schematic Illustration of GGT activation and αvβ3 integrin-targeted delivery of probe 1 for tumor cell NIR fluorescence imaging. (c) The chemical structure of the control probe 1-ctrl.

Fluorescence Imaging of GGT Activity in Live Cells. U87MG, HEK-293 or HUVEC cells (~5 x 104) were seeded to a glass-bottom dish (In Vitro Scientific, D35-20-1-N) and allowed to grow for 24 h. After removal of medium, probe 1 (2 µM) in DMEM (1 mL) was added and incubated at 37 °C for 4 h. To inhibit GGT activity, cells were pretreated with GGsTop (100 µM) for 1 h. To block αvβ3 integrin, cells were pretreated with c-RGD (50 µM) for 1 h. Then probe 1 (2 µM) was added and incubated at 37 °C for another 4 h. Afterwards, cells were co-stained with 0.1 µg/mL Hoechst 33342 for 15 min at r.t.. After replacing the medium, fluorescence images were captured on an Olympus IX73 fluorescent inverted microscope. The fluorescence of mCy-Cl was acquired with ex-

EXPERIMENTAL SECTION Synthesis. The preparation of probes 1 and 1-ctrl is described in the Supporting Information. General Procedure for the Detection of GGT with Probe 1. Probe 1 (5 µM) was dissolved in enzyme reaction buffer (1× PBS buffer, pH = 7.4), and then incubated with GGT (100 U/L) at 37 ºC for varying time. At each time point, the UV-Vis absorption spectra from 400 to 850 nm was acquired, and the fluorescence spectra were recorded from 690 to 850 nm with excitation from 675 to 835 nm by fluorescence synchronous scanning. 2


Page 2 of 9

Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry citation at 650 to 690 nm, and emission from 710 to 750 nm. Hoechst 33342 was excited at 340 to 390 nm, and emission was collected from 420 to 460 nm. For co-localization studies, U87MG cells were incubated with probe 1 (2 µM) for 4 h and then co-stained with 200 nM Lyso-Tracker Red or MitoTracker Green for 20 min at 37 °C. The fluorescence images of the cells were captured with a Leica TCS SP5 confocal laser scanning microscope after replacement with fresh medium. Fluorescence Imaging of GGT in U87MG TumorBearing mice in Real Time. All animal studies were conducted in accordance with institutional animal use and care regulations approved by the Model Animal Research Center of Nanjing University (MARC). Six week-old male nude mice were subcutaneously injected with 1×106 cells to establish U87MG tumor model. Tumors were allowed to grow to 8-10 mm in diameter for imaging studies. Probe 1 or 1-ctrl (25 µM) in 150 µL saline solution was injected through tail vein. To inhibit GGT activity or probe uptake, GGsTop (5 mM) or free c-RGD (2 mM) in 100 uL saline was injected into tumors 1 h before i.v. injection of probe 1. Whole body fluorescence images were acquired using an IVIS Lumina XR III system (excitation, 660; emission, 710 nm) at 0 min, 15 min, 30 min, 1 h, 2 h and 4 h post injection. For ex vivo imaging of GGT in tumor tissue slices, mice was euthanized at 2 h post injection of probe 1 or 1-ctrl, and tumors were excised from mice, frozen at -80 °C. Then, the frozen tumor tissue slices at a thickness of 10 µm were cut using a vibrating-blade microtome, stained with 10 µg/mL Hoechst 33342 at r.t. for 15 min. After washed with PBS three times, fluorescence images of the staining tissues were record on an Olympus IX73 fluorescent inverted microscope.

recognize the c-RGD ligand in probe 1 and promote it binding to the tumor cells. The cell membrane-bounded GGT can then cleave the γ-Glu in probe 1, resulting in highly fluorescent probe 2 that is attached at αvβ3 (red dash line). In process 2, probe 1 can be firstly hydrolyzed and converted into highly fluorescent product 2 by the cell membrane-bound GGT. The αvβ3 receptor on the tumor cell surface can then recognize the c-RGD ligand in probe 2 and promote it binding to the tumor cells (blue dash line). Then, probe 2 can enter into tumor cells via αvβ3 receptor-mediated endocytosis and accumulate in the lysosomes, ultimately producing strong NIR fluorescence in tumor cells. In contrast, neither αvβ3 integrin- nor GGTdeficient normal cells can induce similarly strong intracellular fluorescence. As a result, the combination of dual biomarkerrecognized substrates in probe 1 for both tumor targeted delivery and activation can substantially enhance the selectivity for tumor imaging after systemic administration, which will be very helpful for improved tumor detection in vivo. To substantiate the proposed mechanism, a negative control probe (1ctrl) was also synthesized by esterifying the γ-Glu in probe 1 with an allylic group, thus preventing cleavage by GGT after binding to the tumor cells. Synthesis and Characterization of Probes in Vitro. The synthesis of probes 1 and 1-ctrl was described in Scheme S1. Amine-containing NIR780 fluorophore 4 was synthesized from 2,3,3-trimethylindolenine according to previously reported methods.47,48 After protecting the two free amines in 4 with Boc group, compound 5 was obtained, which was then reacted with 4-chloro-1,2-dihydroxybenzene and followed by deprotection of Boc group to afford the key NIR fluorophore mCyCl (7). Subsequent reaction of compound 7 with Nsuccinimidyl 3-maleimidopropionate yielded compound 8, which was directly reacted with compound 9 to afford compound 10. Conjugation of 10 with commercially available cRGD-SH afforded compound 11. Finally, after removal of Fmoc group, 1-ctrl was obtained, which was followed by deprotection of allylic group to afford probe 1. In order to demonstrate the fluorescent product, we also synthesized the cleaved product 2 by direct coupling of compound 8 with cRGD-SH. The detailed synthesis and characterization data were described in the supporting information. We firstly investigated the response of probe 1 towards GGT by measuring the spectra of both UV-Vis absorption and fluorescence emission upon incubation with GGT in PBS buffer. As shown in Figure 2a, the absorbance peak of 1 at 608 nm gradually decreased while a new absorbance peak at 692 nm appeared concomitantly following treatment with GGT, resulting in an obvious color change from blue to green after 60 min (Figure 2a, insert). Accompanying with the bathochromic shift of UV-Vis absorption, the fluorescence emission at 712 nm also increased with incubation time, with a maximum fluorescence intensity reached after 60 min (Figure 2b). The maximum fluorescence turn-on ratio of probe 1 activated by GGT at 712 nm was calculated to be ~230-fold with excitation from 675 to 835 nm by fluorescence synchronous scanning, which was also demonstrated by a significantly bright fluorescence image (excitation: 660 nm, emission from 700850 nm) of solution 1 following treatment with GGT (Figure 2b, insert). The GGT-triggered conversion of probe 1 to 2 was then monitored using HPLC analysis. Figure 2c showed that 1 (5 µM, retention time, TR = 6.7 min) was efficiently converted

RESULTS AND DISCUSSION Design of αvβ3-Targeting and GGT-Activatable NIR Probe for Cancer Imaging. Figure 1a shows the molecular structure of probe 1, consisting of a GGT-specific substrate γGlu, a self-immolative spacer p-aminobenzyl alcohol (PABA), a NIR fluorophore merocyanine (mCy-Cl), and a αvβ3 integrin receptor-targeting cyclic peptide ligand (c-RGD). The amino acid γ-Glu has been widely used as a GGT-recognition substrate enabling design of specific molecular imaging probes for GGT detection.45,46 c-RGD was introduced to promote the active delivery of probe and entry into the αvβ3-positive tumor cells via an endocytosis process. Moreover, the hydrophilic and negative characteristics of c-RGD can also improve the solubility of probe under physiological conditions and prevent free diffusion into αvβ3-deficient normal tissue cells after activation by GGT. Probe 1 is fluorescence off initially due to the quenching effect of PABA capping at the NIR fluorophore. Upon interaction with GGT, the γ-Glu in probe 1 is cleaved, which subsequently triggers spontaneous elimination of the self-immolative linker PABA, liberating probe 2 with strong NIR fluorescence at 712 nm. Figure 1b illustrates the proposed mechanism of probe 1 for the specific imaging of tumors. After intravenous injection, the small size and hydrophilicity of probe 1 can facilitate the extravasation and penetration into tumor tissues, subsequently becoming fluorescence in the tumors through two plausible processes. In process 1, the αvβ3 integrin overexpressed on the tumor cell surface can firstly 3



to the cleaved product 2 (TR=8.8 min) (Figure 2c), which was also confirmed by MALDI-MS analysis (Figure S1). In contrast, the incubation of 1-ctrl with GGT under the similar conditions could not induce obvious change in neither UV-Vis absorption nor fluorescence emission, which was also verified by HPLC analysis (Figure S2). These results suggested that GGT could efficiently activate probe 1 but not 1-ctrl to yield uncaged product 2, resulting in a remarkable fluorescence enhancement.

tionship (y = 0.205x (U/L) + 0.032), with the correlation coefficient R2 of 0.998 (Figure 3b). The detection limit was calculated to be ~2.91 mU/L (single-to-noise, S/N = 3), which was more sensitive than most of other previously reported GGT detection probes (Table S2). The selectivity of probe 1 towards GGT was next examined by incubating it with GGT, GGT pretreated with its inhibitor GGsTop (100 µM) or other representative proteases in tumor cells, such as Cathepsin B and MMP-2 (Figure 3c). The fluorescence intensity at 720 nm was monitored over the course of incubation using a microplate reader. The results in Figure 3c showed that only GGT could cause a time-dependent enhancement of fluorescence. This enhanced fluorescence was completely inhibited by GGsTop, suggesting that the GGT activity was crucial to turn on the fluorescence of probe 1. In contrast, the fluorescence of probe 1 was not enhanced when incubated with either Cathepsin B or MMP-2. These results indicated that probe 1 was a specific probe for GGT detection.

Figure 2. (a) UV-Vis absorption and (b) fluorescence spectra of probe 1 (5 µM) following incubation with GGT (100 U/L) at 37 ºC for different time. Fluorescence spectra was measured by synchronous fluorescence scanning (λex = 675-835 nm, offset = 15 nm). (c) HPLC analysis of probe 2, probe 1 before (black) and after (red) incubation with GGT (100 U/L) at 37 °C for 60 min. (d) Michaelis-Menten plots of probe 1 with GGT. Error bars represent standard deviation (n = 3).

We then measured the kinetic parameters of 1 toward GGT according to the Michaelis-Menten equation (Figure S3). As shown in Figure 2d, the Michaelis constant Km and the catalytic constant kcat were found to be ~1.85 µM and ~0.00502 s-1, respectively, which could be further used to deduce a kcat/Km value of ~2624 M-1 s-1. All these kinetic values were close to that of our previously reported GGT-responsive NIR probe lacking the c-RGD ligand (kcat/Km = 0.00447 s-1/1.26 µM, Table S1), indicating that the introduction of a tumor-targeting group c-RGD had little effect on the enzymatic cleavage efficiency. Sensitivity and Specificity of Probe 1 toward GGT. Having demonstrated the activation of probe 1 by GGT, the sensitivity for the detection of GGT was then investigated using the optimized reaction conditions. Probe 1 (5 µM) was incubated with varying concentration of GGT (0-100 U/L) in PBS buffer for 60 min, and the resulting fluorescence was acquired. As shown in Figure 3a, probe 1 alone was very stable in PBS buffer, with negligible fluorescence observed after 60 min. Following incubation with GGT, it was clear to found that the fluorescence emission at 712 nm increased possessively with the concentration of GGT, with a plateau reached when the GGT concentration was up to 80 U/L. The plot of normalized fluorescence intensity at 712 nm versus GGT concentration in the range of 0.02−5 U/L showed a great linear correlation rela-

Figure 3. Sensitivity, specificity and stability analysis. (a) Fluorescence spectra of probe 1 (5 µM) following incubation with varying concentration of GGT at 37 ºC for 60 min. (b) Linear fitting curve of fluorescence intensity (λem = 712 nm) with the concentration (c) of GGT from 0.02-5.0 U/L. (c) Time-dependent fluorescence intensity changes of 5 µM probe 1, probe 1 incubated with either GGT (100 U/L), 1.0 µg/mL Cathepsin B, 5 nM MMP-2, or GGT (100 U/L) together with its inhibitor GGsTop (100 µM), 5 µM probe 1-ctrl without or with GGT (100 U/L) (λex/em = 680/720 nm). (d) Fluorescence turn-on ratio (F/F0) of probe 1 (5 µM) following incubation in PBS buffer (pH=7.4) (1, 2), DMEM (3, 4), PBS buffer containing 10% mice serum (5, 6), or DMEM containing 10% FBS (7, 8) in the absence (1, 3, 5, 7) or presence (2, 4, 6, 8) of 100 U/L GGT for 60 min. Error bars represent standard deviation (n = 3).

To further demonstrate the capacity of probe 1 for the detection of GGT activity in complex physiological environment, the stability of 1 in cell culture medium (DMEM), DMEM medium containing 10% FBS or PBS buffer containing 10% mice serum was then tested. There was little fluorescence enhancement after incubation in these conditions, while the addition of GGT could activate probe 1 efficiently and induce similar fluorescence enhancement (Figure 3d). These results re4


Page 4 of 9

Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry vealed that probe 1 was a stable probe capable of detecting GGT activity under complex cell culture or in vivo environment. Fluorescence Imaging of GGT in Live Cells. The ability of probe 1 for the specific imaging of GGT activity was demonstrated in human brain cancer U87MG cells with overexpression of both GGT and αvβ3 integrin (Figure 4a). U87MG cells were incubated with 2 µM probe 1 for different time (0, 1, 2, 4, 6 h), and the resulting intracellular NIR fluorescence was acquired. As shown in Figure 4a, U87MG cells incubated with probe 1 showed punctate intracellular fluorescence, and the intensity increased with the incubation time, with a maximum observed after incubation for over 4 h. The enhanced fluorescence in the cells was attributed to the formation of fluorescence product 2, which was also confirmed by both fluorescence and HPLC analysis of U87MG cells after incubation with probe 1 (Figure S5, Supporting Information). These results suggested that probe 1 could be activated and enter into U87MG cells. In order to reveal the intracellular distribution of fluorescence, colocalization study of probe 1-treated U87MG cells with lysosomal tracker (Lyso-Traker Red) and mitochondrial tracker (MitoTracker Green) was then carried out to show that the punctuate intracellular NIR fluorescence (pseudo red color) overlaid well with the fluorescence from the Lyso-Traker Red (pseudo green color, Figure 4b). In contrast, there was poor colocalization with the Mito-Tracker Green. This indicated that the fluorescent product 2 accumulated mainly in the lysosomes, presumably due to selective cellular uptake via the αvβ3 integrin receptor-mediated endocytosis.

GGT inhibitor (GGsTop, 100 µM) for 1 h, and then incubated with 2 µM probe 1 for another 4 h. The fluorescence in U87MG cells was then acquired. As shown in Figure 5a, U87MG cells incubated with probe 1 showed strong intracellular fluorescence, which could be efficiently suppressed by GGsTop, suggesting that the fluorescence enhancement in U87MG cells was selectively induced by GGT. To demonstrate that the enhanced intracellular fluorescence was ascribed to the αvβ3 integrin-mediated cellular uptake of fluorescent product 2, a blocking study with c-RGD (50 µM) was next carried out. Figure 5a showed negligible intracellular fluorescence in U87MG cells blocked with c-RGD, suggesting that the ligand c-RGD played an important role in cellular uptake of GGT-cleaved fluorescent product. Moreover, to further demonstrate the capacity of probe 1 for differentiating tumor cells from normal cells, GGT-deficient normal human umbilical vein endothelial HUVEC cells and GGT-positive but αvβ3 integrin receptor-deficient human embryonic kidney HEK293 cells were chosen for the next study. The relative activity of GGT in these three different cell lines were firstly evaluated using a commercial GGT substrate (AMC-Glu), showing that the GGT activity in HEK293 cells was a little higher than that of U87MG cells, while HUVEC cells exhibited very low GGT activity (Figure S6). We then incubated these cells with probe 1 under the same conditions, and the resulting images revealed that neither HUVEC nor HEK293 normal cells showed obvious intracellular NIR fluorescence as compared to that of U87MG tumor cells. In contrast, the incubation of HEK293 cells with our previously reported probe GANP lacking the cRGD ligand showed higher intracellular fluorescence than that of U87MG cells (Figure S7). These results suggested that probe 1 was more feasible to differentiate U87MG tumor cells from HEK293 cells in terms of significantly different intracellular fluorescence, confirming the high capacity of 1 for selective detection of GGT in tumor cells. To the best of our knowledge, this is the first GGT-activatable fluorescence probe capable of distinguishing GGT-positive tumor cells from GGT-positive normal cells (e.g., HEK293). Fluorescence Imaging of GGT in Xenograft U87MG Tumors. To evaluate the capacity of probe 1 for non-invasive fluorescence imaging of GGT in vivo, probe 1 (25 µM) in saline (150 µL) was i.v. injected into U87MG tumor-bearing mice. The whole-body fluorescence was monitored longitudinally using an IVIS fluorescence imager. As shown in Figure 6a, mice injected with probe 1 showed gradually increased NIR fluorescence in the U87MG tumors, with the maximum fluorescence observed at 2 h. This strong tumor fluorescence could be efficiently reduced when the mice received intratumoral injection of GGsTop (5 mM), which was found to be ~3-fold lower than that induced by probe 1 at 2 h (Figure 6b). The tumor-to-background (TBR) ratio of mice treated with probe 1 reached ~2.1 at 4 h, which was significantly higher than that in mice treated with i.v. injection of GGT-resistant 1ctrl (~1.4) (Figure S7). These results suggested that probe 1 could be efficiently delivered into tumor tissue and selectively activated by GGT, resulting in bright fluorescence in tumors capable of discriminating tumors from normal tissues. To further examine whether the c-RGD ligand in probe 1 also played a key role in enhancing tumor fluorescence in living mice, free c-RGD (2 mM) was directly injected into the tumor to block the αvβ3 integrin receptors, and probe 1 was i.v. injected 1 h

Figure 4. Fluorescence imaging of U87MG cells incubated with probe 1. (a) Fluorescence images of U87MG cells following incubation with probe 1 (2 µM) for 0, 1, 2, 4, and 6 h. (b) Fluorescence images of probe 1-treated U87MG cells following staining with either Lyso-tracker Red or Mito-tracker Green. Cells were incubated with probe 1 (2 µM) for 4 h and then co-stained with 200 nM Lyso-Tracker Red or Mito-Tracker Green for 20 min.

To demonstrate the specificity of probe 1 for GGT imaging in live cells, we pretreated U87MG cells with the effective 5



later. The results showed that both the tumor fluorescence and TBR were also significantly reduced when the αvβ3 was blocked with free c- RGD (Figures 6a, b, & S7). This lower tumor fluorescence could be due to the remarkably reduced delivery of probe 1 into tumor tissues, thus limiting its activation and the subsequent uptake of fluorescent product 2 into tumor cells. Moreover, the significantly different tumor fluorescence in Figure 6a was also confirmed by the ex vivo imaging of tumor tissue slices resected 2 h after each treatment, showing that mice injection with probe 1 alone exhibited remarkably bright fluorescence compared to other three control

groups (Figure 6c). These findings demonstrated that probe 1 was an effective molecular probe allowing for non-invasive imaging of GGT in tumors in real time, which could be further applied to specifically differentiate tumor tissues from normal tissues in vivo. However, Figure 6a also showed strong fluorescence in the livers of mice receiving either i.v. injection of probe 1, which was also found in healthy mice (Figure S8). This enhanced liver fluorescence could be due to the nonspecific activation of probe 1 in the livers that are reported to show high GGT activity,43,44 implying that probe 1 may not be suitable for non-invasive imaging liver tumors in living mice.

Figure 5. (a) Fluorescence images of U87MG cells incubated with 2 µM probe 1, probe 1 with GGT inhibitor GGsTop (100 µM) or free cRGD (50 µM). (b) Fluorescence images of U87MG tumor cells, HEK293 normal cells or HUVEC normal cells following incubation with probe 1 (2 µM) for 4 h. Nucleus were co-stained with Hoechst 33342 (0.1 µg/mL). The blue fluorescence was acquired from 420 nm–460 nm upon excitation at DAPI channel, and the red fluorescence was acquired from 710 nm–750 nm upon excitation at 680 nm.

Figure 6. Fluorescence imaging of GGT in vivo. (a) Real-time fluorescence images of U87MG tumor-bearing mice receiving i.v. injection of probe 1 (25 µM, 150 µL), 1-ctrl (25 µM, 150 µL), probe 1 with i.t. injection of GGsTop (5 mM, 100 µL), or probe 1 with i.t. injection of free c-RGD (2 mM, 100 µL). Red arrows indicate the location of tumors. (b) Quantitative analysis of the average fluorescence intensity of tumor as indicated in (a). Values are mean ± SD (n = 3). ** P < 0.01, *** P < 0.001. (c) Fluorescence images of tumor tissue slices resected from U87MG tumor-bearing mice 2h after i.v. injection of probe 1 or probe 1-ctrl, and then stained with DAPI.

In conclusion, we have combined the mechanism of receptor-mediated cellular uptake with molecular target-triggered

CONCLUSIONS 6


Page 6 of 9

Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry H.; Kim, H. M. Anal. Chem. 2014, 86, 5638-5641. (8) Zhang, R.; Sun, Y.; Tian, M.; Zhang, G.; Feng, R.; Li, X.; Guo, L.; Yu, X.; Sun, J. Z.; He, X. Anal. Chem. 2017, 89, 6575-6582. (9) Uddin, M. J.; Crews, B. C.; Blobaum, A. L.; Kingsley, P. J.; Gorden, D. L.; Mcintyre, J. O.; Matrisian, L. M.; Subbaramaiah, K.; Dannenberg, A. J.; Piston, D. W. Cancer Res. 2010, 70, 3618-3627. (10) Levi, J.; Cheng, Z.; Gheysens, O.; Patel, M.; Chan, C. T.; Wang, Y.; Namavari, M.; Gambhir, S. S. Bioconjugate Chem. 2007, 18, 628-634. (11) Gao, M.; Yu, F.; Lv, C.; Choo, J.; Chen, L. Chem. Soc. Rev. 2017, 46, 2237-2271. (12) Wang, Y.; Li, J.; Feng, L.; Yu, J.; Zhang, Y.; Ye, D.; Chen, H. Anal. Chem. 2016, 88, 12403-12410. (13) Zheng, M.; Wang, Y.; Shi, H.; Hu, Y.; Feng, L.; Luo, Z.; Zhou, M.; He, J.; Zhou, Z.; Zhang, Y. Ye, D. ACS Nano 2016, 10, 10075-10085. (14) Thomas, J. A. Chem. Soc. Rev. 2015, 44, 4494-4500. (15) Kim, C.; Lee, D.; Kim, C.; Lee, K.; Lee, C.; Ahn, I. Anal. Chem. 2014, 86, 3825-3833. (16) Xie, C.; Upputuri, P. K.; Zhen, X.; Pramanik, M.; Pu, K. Biomaterials 2017, 119, 1-8. (17) Kamb, A.; Wee, S.; Lengauer, C. Nat. Rev. Drug. Disc. 2007, 6, 115-120. (18) Peng, H.; Chen, B.; Huang, W.; Tang, Y.; Jiang, Y.; Zhang, W.; Huang, Y. Nano Lett. 2017, 17, 7684-7690. (19) Qian, Y.; Qiao, S.; Dai, Y.; Xu, G.; Dai, B.; Lu, L.; Yu, X.; Luo, Q.; Zhang, Z. ACS Nano 2017, 11, 9536-9549. (20) Chen, D.; Li, B.; Cai, S.; Wang, P.; Peng, S.; Sheng, Y.; He, Y.; Gu, Y.; Chen, H. Biomaterials 2016, 100, 1-16. (21) Xia, M.-C.; Cai, L.; Zhang, S.; Zhang, X. Anal. Chem. 2017, 89, 1238-1243. (22) Tian, X.; Baek, K.; Shin, I. Chem. Sci. 2013, 4, 947-956. (23) Sun, X.; Du, R.; Zhang, L.; Zhang, G.; Zheng, X.; Qian, J.; Tian, X.; Zhou, J.; He, J.; Wang, Y.; Wu, Y.; Zhong, K.; Cai, D.; Zou, D.; Wu, Z. ACS Nano 2017, 11, 7049-7059. (24) Hu, J.; Liu, L.; Li, Z.; Zhuo, R.; Zhang, X. J. Mater. Chem. B 2016, 4, 1932-1940. (25) Wang, T.; Wang, D.; Liu, J.; Feng, B.; Zhou, F.; Zhang, H.; Zhou, L.; Yin, Q.; Zhang, Z.; Cao, Z.; Yu, H.; Li, Y. Nano Lett. 2017, 17, 5429-5436. (26) Shim, M. K.; Yoon, H. Y.; Ryu, J. H.; Koo, H.; Lee, S.; Park, J. H.; Kim, J.; Lee, S.; Pomper, M. G.; Kwon, I. C. Angew. Chem. Int. Ed. 2016, 55, 14698-14703. (27) Jiang, Y.; Pu, K. Small, 2017, 13, 1700710. (28) Lee, H.; Kim, J.; Kim, H.; Kim, Y.; Choi, Y. Chem. Commun. 2014, 50, 7507-7510. (29) Kim, J.; Kim, H.; Choi, Y. J. Mater. Chem. B 2017, DOI: 10.1039/c7tb02180a. (30) Kim, J.; Won, Y.; Goh, S.; Choi, Y. J. Mater. Chem. B 2016, 4, 6787-6790. (31) Fraser, A.; Ebrahim, S.; Smith, G. D.; Lawlor, D. A. Hepatology 2007, 46, 158-165. (32) Kushwaha, N.; Srivastava, S. Antonie van Leeuwenhoek 2014, 105, 45-56. (33) De Oliveira, L. P. M.; De Jesus, R. P.; Boulhosa, R. S. S. B.; Onofre, T.; Mendes, C. M. C.; Vinhas, L.; Waitzberg, D. L.; Lemaire, D. C.; Cavalcante, L. N.; Lyra, A. C. J. Am. Coll. Nutr. 2016, 35, 436442. (34) Corti, A.; Franzini, M.; Paolicchi, A.; Pompella, A. Anticancer Res. 2010, 30, 1169-1181. (35) Urano, Y.; Sakabe, M.; Kosaka, N.; Ogawa, M.; Mitsunaga, M.; Asanuma, D.; Kamiya, M.; Young, M. R.; Nagano, T.; Choyke, P. L. Sci. Transl.Med. 2011, 3, 110-119. (36) Li, L.; Shi, W.; Wu, X.; Gong, Q.; Li, X.; Ma, H. Biosens. Bioelectron. 2016, 81, 395-400. (37) Zhang, P.; Jiang, X.; Nie, X.; Huang, Y.; Zeng, F.; Xia, X.; Wu, S. Biomaterials 2016, 80, 46-56. (38) Wang, F.; Zhu, Y.; Zhou, L.; Pan, L.; Cui, Z.; Fei, Q.; Luo, S.; Pan, D.; Huang, Q.; Wang, R.; Zhao, C.; Tian, H.; Fan, C. Angew.

activation of fluorescence to develop a tumor-targeting and GGT-activatable NIR fluorescence probe (1) for selective cancer imaging in living mice. We showed that probe 1 could be efficiently converted into the highly fluorescent product 2 upon interaction with GGT, resulting in a remarkable ~230fold enhancement of NIR fluorescence (λem = 712 nm). Cell studies showed that probe 1 could induce bright NIR fluorescence distributed mainly in the lysosome of U87MG tumor cells, which was not observed in the GGT-positive but αvβ3deficient normal cells. We also demonstrated that probe 1 could readily extravasate and enter into tumor tissues through αvβ3 integrin receptor-mediated active delivery after i.v. injection into mice, producing strong tumor fluorescence that could be inhibited by either GGsTop or free c-RGD. These results suggested that probe 1 with the integration of αvβ3-targeting ligand c-RGD and GGT-cleavable substrate γ-Glu was an effective molecular probe with improved selectivity for tumor imaging in vivo. With this similar principle, it is feasible to develop other dual-targeting imaging probes for the noninvasive detection of essential biological processes by using different receptor-targeting groups (e.g., folic acid, aptamer and antibody) and/or different biomolecule-recognitive substrates, which could facilitate to improve diagnosis and treatment of diseases.

ASSOCIATED CONTENT Supporting Information Supplementary Figures, synthetic procedures, detailed experimental operation and NMR, HRMS-characterization. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Authors * Tel/Fax: +86-510-85514422; E-mail: [email protected]. * Tel/Fax: +86-25-89681905; E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial supports from the National Natural Science Foundation of China (21775071, 21505070, 21327902, 21632008 and 21371082), Natural Science Foundation of Jiangsu Province (BK20150567) and the Foundation Research Funds for the Central Universities (020514380096) were acknowledged.

REFERENCES (1) Razgulin, A.; Ma, N.; Rao, J. Chem. Soc. Rev. 2011, 40, 4186-4216. (2) Lyu, Y.; Pu, K. Adv. Sci. 2017, 1600481. (3) Yan, R.; Ye, D. Sci. Bull. 2016, 61, 1672-1679. (4) Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.; Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2014, 136, 14136-14142. (5) Luo, Y.; Zhang, L.; Liu, W.; Yu, Y.; Tian, Y. Angew. Chem. Int. Ed. 2015, 54, 14053-14056. (6) Ye, D.; Shuhendler, A. J.; Cui, L.; Tong, L.; Tee, S. S.; Tikhomirov, G. A.; Felsher, D. W.; Rao, J. Nat. Chem. 2014, 6, 519526. (7) Sarkar, A. R.; Heo, C. H.; Lee, H. W.; Park, K. H.; Suh, Y.

7



Chem. Int. Ed. 2015, 54, 7349-7353. (39) Tong, H.; Zheng, Y.; Zhou, L.; Li, X.; Qian, R.; Wang, R.; Zhao, J.; Lou, K.; Wang, W. Anal. Chem. 2016, 88, 10816-10820. (40) Li, L.; Shi, W.; Wang, Z.; Gong, Q.; Ma, H. Anal. Chem. 2015, 87, 8353-8359. (41) Park, S.; Lim, S.-Y.; Bae, S. M.; Kim, S.-Y.; Myung, S.-J.; Kim, H.-J. ACS Sens. 2016, 1, 579-583. (42) Luo, Z.; Feng, L.; An, R.; Duan, G.; Yan, R.; Shi, H.; He, J.; Zhou, Z.; Ji, C.; Chen, H.-Y.; Ye, D. Chem. Eur. J. 2017, 23, 1477814785. (43) Braun, J. P.; Rico, A. G.; Benard, P.; Thouvenot, J. P.; Bonnefis, M. J. J. Dairy Sci. 1977, 60, 1283-1287.

(44) Mason, J. E.; Starke, R. D.; Van Kirk, J. E. Prev. Cardiol. 2010, 13, 36-41. (45) Orlowski, M.; Meister, A. Biochim. Biophys. Acta. 1963, 73, 679-681. (46) I. N. White, N. Razvi, R. M. Lawrence, M. M. Manson, Anal. Biochem. 1996, 233, 71 –75. (47) Bindman, N. A.; Der Donk, W. A. V. J. Am. Chem. Soc. 2013, 135, 10362-10371. (48) Kang, N.; Park, S.; Ang, X. W. E.; Samanta, A.; Driessen, W. H. P.; Ntziachristos, V.; Vasquez, K. O.; Peterson, J. D.; Yun, S.; Chang, Y. Chem. Commun. 2014, 50, 6589-6591.

8


Page 8 of 9

Page 9 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

For Table of Contents Only


9

Targeted Delivery of a Î³-Glutamyl Transpeptidase ... - ACS Publications

Recommend Documents