An activatable near-infrared fluorescent probe for dipeptidyl peptidase

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An activatable near-infrared fluorescent probe for dipeptidyl peptidase IV and its bioimaging applications in living cells and animals Tao Liu, Jing Ning, Bo Wang, Bin Dong, Song Li, Xiangge Tian, Zhenlong Yu, Yulin Peng, Chao Wang, Xinyu Zhao, Xiaokui Huo, Cheng-Peng Sun, Jing-Nan Cui, Lei Feng, and Xiaochi Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04957 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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Analytical Chemistry

An activatable near-infrared fluorescent probe for dipeptidyl peptidase IV and its bioimaging applications in living cells and animals

Tao Liua,b,‡, Jing Ninga,‡, Bo Wanga, Bin Dongc, Song Lia, Xiangge Tiana, Zhenlong Yua, Yulin Penga, Chao Wanga, Xinyu Zhaoa, Xiaokui Huoa, Chengpeng Suna, Jingnan Cuib, Lei Fenga,b,*, and Xiaochi Maa,*

a

College of Pharmacy, Academy of Integrative Medicine, Dalian Medical University, Lvshun South Road No 9, Dalian 116044, China;

b

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China;

c

School of Physics and Materials Engineering, Dalian Nationalities University, 18 Liaohe West Road, Dalian 116600, China.

‡ These two authors equally contributed to the present work.

∗Address for correspondence: Xiaochi Ma & Lei Feng, College of Pharmacy, Research Institute of Integrated Traditional and Western Medicine, Dalian Medical University, Western 9 Lvshun south road, Dalian, 116044, China. Tel/Fax: +86-411-86110419. E-mail: [email protected] & [email protected]

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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

ABSTRACT Visualization of endogenous disease-associated enzymes is of great clinical significance, as it could allow earlier clinical diagnosis and timely intervention. Herein, we first synthesized and characterized an enzyme-activatable near-infrared fluorescent probe, GP-DM, for determining the activity of dipeptidyl peptidase IV (DPP IV), which is associated with various pathological processes, especially in diabetes and malignant tumors. GP-DM emitted significant turn-on NIR fluorescent signals simultaneously in response to DPP IV, making it favorable for accurately and dynamically monitoring DPP IV activity in vitro and in vivo. GP-DM exhibited excellent specificity and sensitivity in DPP IV imaging, as indicated by its higher catalytic activity than other human serine hydrolases and by its strong anti-interference ability to a complex biological matrix, which was fully characterized in a series of phenotyping reactions and inhibition assays. Encouraged by the advantages mentioned above, we successfully used GP-DM to evaluate endogenous DPP IV activity in various biological samples (plasma and tissue preparations) and living tumor cells, and performed real-time in vivo bioimaging of DPP IV in zebrafish and tumor-bearing nude mice. All of the results reflected and highlighted the potential application value of GP-DM in the early detection of pathologies, individual tailoring of drug therapy, and image-guided tumor resection. Furthermore, our results revealed that DPP IV, a key target enzyme, is closely associated with the migration and proliferation of cancer cells, and regulating the biological activity of DPP IV may be a useful approach for cancer therapy. Keywords: Dipeptidyl peptidase IV, Near-infrared fluorescent probe, Enzymatic activity, In vivo imaging, In vitro imaging


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INTRODUCTION The ability to measure key biochemical and molecular processes of a specific disease represents a potentially powerful tool for early diagnosis and timely clinical intervention. Recently, the visualization of changes in the activity of metabolising enzymes that are disease selective has been developed and urgently pursued.1-3 However, during the onset and progression of diseases, the complexity of the dynamic living system makes it difficult to track and visualize the actual in vivo functional level of the specific enzyme. Therefore, much efforts have been devoted to targeting and treating key disease-associated enzymes with fluorescent probes to visualize their activities for clinical diagnosis.4-7 Dipeptidyl peptidase IV (DPP IV) is a transmembrane glycoprotein with proteolytic activity capable of cleaving N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position.8-11 DPP IV is responsible for the degradation of a number of biological

peptides,

including

glucagon-like

polypeptide

and

glucose-dependent

insulinotropic polypeptide, that play an essential role in maintaining glucose homeostasis.12-14 As an effective treatment for type 2 diabetes, DPP IV inhibition exerts positive effects by degrading incretins, and control of blood glucose levels which has been demonstrated in biological models and in clinical trials.15-17 Thus, the specific functional evaluation of DPP IV for the precision therapy of diabetes is an active area of research.18,19 Moreover, DPP IV is frequently dysregulated in cancer, providing a link to the impairment of glucose homeostasis in cancer cells.20 DPP IV is a multifunctional protein potentially associated with progression and metastatic spread, promoting it as a vital therapeutic target to treat malignancy.21-23 An ever-increasing body of evidence has suggested that alterations of plasma DPP IV level, and



its monitoring may provide guidance for early diagnosis or prognosis assessment of many metabolic disorders, including diabetic kidney disease, lung diseases, cholestasis, hepatocirrhosis, hepatic fibrosis and non-alcoholic fatty liver.24-27 Therefore, accurate measurement and efficient imaging of DPP IV is of great importance for DPP IV dysfunction-related disease diagnosis, drug discovery and clinical practice. During the past decade, several fluorescent probes have been reported to detect DPP IV in tissue and cell samples.19,28-31 However, some of DPP IV sensors do not exhibit sufficient specificity to precisely measure its real activity in complex biosamples. Most importantly, the in vivo applications of the reported DPP IV fluorescent sensors are limited due to the autofluorescence interference of biological matrix and poor tissue penetration capacity of sensors. Imaging reporters that emit near-infrared (NIR) optical signals have enabled minimization of tissue autofluorescence and light scattering, as well as improved photon penetration through tissue, thereby enabling more accurate imaging or functional evaluation, even in living animals.32-36 Herein, to develop a novel NIR fluorescent probe for sensitive and specific measurement of DPP IV in various biological systems is urgently demanded. In the present work, we first synthesized and characterized a NIR fluorescent probe, GP-DM, that exhibited good specificity, sensitivity and practicability for DPP IV imaging, as indicated by its substrate selectivity and catalytic characteristic for DPP IV. By virtue of its advantages, GP-DM was used to monitor DPP IV in various human tissues, living cells and zebrafish. Additionally, GP-DM can provide accurate and dynamic images of the DPP IV functional level, and these findings all suggest the great potential for application of this NIR probe in in vivo fluorescence bioimaging.


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EXPERIMENTAL SECTION Materials and instruments. Dipeptidyl peptidases, including DPP IV and DPP VIII, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), human serum albumin (HSA), leucine aminopeptidase (LAP), and prolidase (PAP) were obtained from Sigma-Aldrich (St. Louis, USA). Recombinant carboxylesterases, including human carboxylesterase 1 (hCE1b, hCE1c) and human carboxylesterase 2 (hCE2), were obtained from BD Biosciences (New Jersey, USA). Pepsin, lysozyme, lipase and sitagliptinon were purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). Pooled human liver microsomes (HLM), kidney microsomes (HKM) and intestinal microsomes (HIM) were purchased from the Rild Research Institute for Liver Diseases (Shanghai, China). Carbonic anhydrase (CA) was obtained from Worthington Biochemical Corporation (Lakewood, USA). Fibroblast activation protein (FAP) was purchased from R&D System (MN, USA). Trypsin was purchased from Solarbio (Beijing, China), and proteinase K was supplied by AMRESCO (Texas, USA). The human HepG2 and LoVo cell lines were obtained from ATCC (Manassas, VA). Small interfering RNAs (siRNA) against human DPP IV were obtained from Shanghai GenePharma Co (Shanghai, China). Lipofectamine 2000 Reagent was purchased from Invitrogen (NY, USA). A DPP IV ELISA Kit was obtained from Shanghai Lengton Bioscience (Shanghai, China). All other chemicals and reagents were analytical grade or the highest quality commercial grade. 1

H NMR and

13

C NMR spectra were recorded using a Bruker Avance II (500 MHz).

Accurate mass detection was performed using a Hybrid Ion Trap-Orbitrap Mass Spectrometer (LTQ Orbitrap XL, Thermo). Absorption spectra and fluorescence emission/excitation spectra



were measured on a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek).

Synthesis of GP-DM. The detailed synthesis and structural characterization of GP-DM are described in the supplementary data (Supplementary Scheme S1, Figure S1-S3).

General procedures for DPP IV activity assay. All DPP IV activity assays were performed in 100 mM phosphate buffer (PBS) with pH value of 7.4. Incubation systems were consisted of PBS and recombinant DPP IV, microsomes or plasma, with a total reaction volume of 200 µL. Then, GP-DM was added to reaction system for starting the reactions. After shaking incubation at 37 °C for a certain time, 200 µL acetonitrile was used for terminating hydrolysis mediated by DPP IV. The mixtures were then centrifuged at 20,000 g for 10 min, and supernatant were subsequently taken for fluorescence analysis. To verify that the reaction was enzyme dependent, samples without enzyme sources were also carried out. The analysis in the present study was carried out in duplicate. To explore the applicability of GP-DM for DPP IV detection, the selectivity of this probe was further investigated by using other hydrolases, including proteinase K (10 µg/ml), carbonic anhydrase (CA, 10 µg/ml), pepsin (10 µg/ml), butyrylcholinesterase (BChE, 10 µg/ml), acetylcholinesterase (AChE, 10 µg/ml), human serum albumin (HSA, 500 µg/ml), lysozyme (10 µg/ml), trypsin (10 µg/ml), lipase (10 µg/ml), human carboxylesterases (CES; 10 µg/ml), and other human prolyl-cleaving enzymes, including DPP VIII (1 µg/ml), leucine aminopeptidase (LAP, 1 µg/ml), prolidase (PAP, 1 µg/ml), and FAP (1 µg/ml).

Evaluating DPP IV in human tissue preparations and plasma. In order to assess the applicability of GP-DM for DPP IV detection in complicated biosamples, the formation rate


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of ADM (metabolite of GP-DM) was used as an indicator of DPP IV activity in various tissue microsomes (HIM, HKM, and HLM), as well as human plasma.

DPP IV inhibition assay. A mixture of GP-DM (100 µM), the specific DPP IV inhibitor sitagliptin (0-0.5 µM), human kidney microsomes (20 µg/mL) or human intestine microsomes (50 µg/mL) or recombinant human DPP IV (1 µg/mL) in PBS, was incubated for 20 min at 37 °C. The IC50 values were determined by incubating GP-DM with a various concentrations of sitagliptin. The inhibitory effects were indicated as the percent residual fluorescent intensities.

Cell culture and cell bioimage experiment. HepG2 or LoVo cells were cultured in MEM/EBSS supplemented with 10% FBS in a 5% CO2 at 37 °C atmosphere. Cells were seeded at a density of 1 × 105 cells per dish and allowed to adhere for 12 h. The adherent cells were washed twice with FBS-free culture medium. The stock solution of probe GP-DM (50 mM) in DMSO was diluted into the FBS free media to a specific final concentration. The cells were then incubated at 37 °C for 60 min before imaging on under a confocal microscope (Leica SP8, Germany). The excitation wavelength was set at 458 nm, and fluorescence emission windows of 630-690 nm were used.

siRNA design and transfection. Three small interfering (si) RNAs against human DPP IV were designed by Shanghai GenePharma Co, with the following sequences: siRNA#1: 5'-AUA UGC CAA UUU AUG ACC CTT-3', siRNA#2: 5'-AUU GAG GUU ACG UAC CCU CTT-3', and siRNA#3: 5'-AUA UGU UGG UGU GCU GUG CTT-3'. Cells were transiently transfected with DPP IV siRNAs using Lipofectamine 2000 Reagent, followed by Western



blotting or other experiments.

Cell Migration Assay. A scratch assay (wound-healing assay) was performed to evaluate cell migration. Briefly, LoVo cells, transfected with control siRNA and DPP IV siRNA for 24 h, were re-plated in six-well plates to grow to full confluence and wounded with a sterile 100 mL pipette tip after 6 h of serum starvation. Then, the plates were washed to remove detached cells. After 48 h of incubation, the cells were stained with 10 µM GP-DM at 37 °C for 30 min. After washing with phosphate-buffered saline (PBS) buffer, the wound gap was observed using a confocal microscope (Leica SP8, Germany).

Colony Formation Assay. To evaluate the in vitro practicability of GP-DM, we also performed a colony formation assay. Briefly, LoVo cells, transfected with control siRNA and DPP IV siRNA for 24 h, were seeded in six-well plates (1.0 × 103 per well) containing 2 mL growth medium with 10% FBS and cultured for 14 days, allowing viable cells to grow into macroscopic colonies. The colonies were stained with 10 µM GP-DM at 37 °C for 30 min; then, the cells were imaged using a confocal microscope (Leica SP8, Germany).

In vivo imaging of DPP IV in tumor-bearing mice. All protocols for this animal study conformed to the Guide for the Care and Use of Laboratory Animals. The animal studies were approved by the ethics committee of Dalian Medical University and performed in strict accordance with relevant guidelines. HepG2 and LoVo cells (5×106 cells) suspended in PBS (100 µL) were subcutaneously injected into the left and right flanks of athymic nude mice (20 ± 2 g), respectively. When the tumors reached a size of approximately 4 × 5 mm in diameter, GP-DM (200 µM in PBS) was subcutaneously injected into the tumors.


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In vivo NIR fluorescence tomographic images were acquired with an in vivo spectrum imaging system. Imaging was performed at a predetermined post-injection time point. The peak excitation wavelength was 488 nm, and multi-spectral imaging was performed from 670 to 730 nm (NightOWL II LB983, Berthold).

In vivo imaging of DPP IV in zebrafish. Zebrafishes were maintained in E3 embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, and 5–10% methylene blue; pH = 7.4). In fluorescence imaging experiments, one-, three-, five- and seven-day-old zebrafishes were incubated with 5 µM GP-DM in E3 embryo medium for 60 min at 28 °C. After washing with PBS to clean up the remaining GP-DM, the fishes were imaged using a confocal microscope (Olympus FV1000, Japan). To verify that the fluorescence signal was probe-dependent, control incubations were also performed under same reaction conditions without GP-DM.

RESULTS AND DISCUSSION Synthesis and Spectral Characterization of GP-DM. With its combination of specificity for the substrate and a catalytic preference for hydrolyzing substrates with glycine at the terminal position and C-2 of proline,28,29 the probe GP-DM was designed by the introduction of a glycylprolyl (Gly-Pro) group into ADM via amidation (Scheme 1). The detailed synthetic procedure for GP-DM is provided in Scheme S1, and the chemical structure of GP-DM was identified by 1H-, 13C-NMR and HRMS. (Supplementary Figure S1-S3).



Scheme 1. Proposed sensing mechanism for DPP IV enzymatic activation of GP-DM. ADM can be released by DPP IV-mediated amide bond-cleavage of GP-DM. Upon the addition of DPP IV, the amide bond of GP-DM was easily cleaved and released ADM (Supplementary Figure S4), which triggered a remarkable NIR fluorescence enhancement with a peak at 658 nm (Figure 1). Additionally, as shown in Figure 1, an excellent linear correlation (R2 = 0.992, Figure 1) between the fluorescence intensities and DPP IV concentrations from 0 to 1.5 µg/mL was observed. The high sensitivity of GP-DM could be ascribed to the little dye emission interference of matrix autofluorescence. Thus, GP-DM could sever as a “Switch-ON” NIR fluorescent probe for DPP IV. Upon treatment with DPP IV, a remarkable change in UV absorption profile of GP-DM was also observed, with the appearance of a new band peak at 458 nm and simultaneously resulting in the disappearance of the absorption peak at 410 nm of GP-DM (Supplementary Figure S5). The color change from yellow to pink allows the colorimetric detection of DPP IV using the naked eye.

We also investigated the effects of varied pH on the fluorescence response of GP-DM and its metabolite ADM (Supplementary Figure S6). In fact, GP-DM was extremely weak


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fluorescence in a wide pH range, indicating the high stability and low background fluorescence of GP-DM under varied pH condition. In contrast, the obvious fluorescence off-on response could be always observed in pH range of 4-12. Thus, GP-DM could work well at physiological pH, and PBS was controlled at pH 7.4 for further investigation.

Figure 1. a) Fluorescence spectra of GP-DM (10 µM) upon the addition of increasing concentrations of DPP IV (0-4 µg/mL) in acetonitrile-phosphate buffer (v/v = 1:1, pH 7.4) at 37 °C for 30 min. λex = 458 nm. b) Fluorescence intensity of GP-DM upon the addition of increasing concentrations of DPP IV (0-4 µg/mL) in acetonitrile-phosphate buffer (v/v = 1:1, pH 7.4) at 37 °C for 30 min. Selectivity of GP-DM. To explore the applicability of GP-DM for sensing endogenous DPP IV in biological samples, the specificity of our sensor was explored (Fig.2). An obvious fluorescence enhancement was realized with GP-DM in the present of DPP IV (1 µg/mL), while no distinct fluorescence signal was emitted from the incubation samples in the present of other common hydrolases, including human serum albumin (HSA), butyrylcholinesterase (BChE), acetylcholinesterase (AChE), human carboxylesterases (CES1b, CES1c and CEs2), carbonic anhydrase (CA), trypsin, pepsin, proteinase K, lysozyme, lipase, and even some human prolyl-cleaving peptidases, including DPP VIII, FAP, prolidase (PAP) and leucine aminopeptidase (LAP).



Figure 2. Fluorescence response of GP-DM (10 µM) at 658 nm (λex = 458 nm) upon the addition of various human hydrolases or proteins. The fluorescence excitation spectra were detected in acetonitrile-phosphate buffer (v/v = 1:1, pH 7.4) at 37 °C for 60 min. To further investigate the selectivity of GP-DM for DPP IV in complicated biosamples, inhibition studies were performed, by using sitagliptin, a selective inhibitor of DPP IV.15 As shown in the supplementary data (Figure S7), sitagliptin could strongly inhibit GP-DM hydrolysis in HIMs and HKMs; a similar inhibitory tendency and IC50 values of sitagliptin against DPP IV were observed for recombinant DPP IV, HIM and HKM. These above-mentioned results fully confirmed that human tissue preparations could be served to high-throughput screen DPP IV inhibitor for replacing expensive recombinant DPP IV, due to the preferred anti-interference ability of GP-DM in a complex biological system. Additionally, our findings strongly suggested that GP-DM hydrolysis was specifically hydrolyzed by DPP IV, implying our sensor was an efficient tool for DPP IV activity assay in human samples.

Enzymatic kinetics of DPP IV-mediated GP-DM hydrolysis. In this study, the kinetics of GP-DM hydrolysis was well-characterized with various enzyme sources, such as human


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recombinant DPP IV, HIM and HKM. As shown in Table S1 and Figure S8, the GP-DM hydrolysis in recombinant human DPP IV and tissue preparations displayed similar properties, including kinetic parameters and substrate inhibition kinetic plots. Notably, the Km values for GP-DM hydrolysis determined in HIM and HKM were agreed well with that determined in recombinant human DPP IV, indicating a predominant role of DPP IV for GP-DM hydrolysis in various human tissues. These results provide additional evidence confirming the excellent selectivity and accuracy of GP-DM towards DPP IV through quantitative measurement of the enzymatic activity of DPP IV in different biological samples.

Quantification of DPP IV in human samples. Recently, DPP IV has received more attention due to its association with tumors, diabetes and other serious diseases endangering human health.22,37-39 A substantial body of research has demonstrated links between DPP IV expression level variation and many diseases, implying DPP IV was a potential biomarker for the early disease diagnosis and treatment.24-26 Therefore, accurate measurement of DPP IV is of great importance for DPP IV dysfunction-related disease diagnosis and treatment, drug development and clinical usage. Subsequently, the applicability of GP-DM for the quantitative determination of DPP IV activities in human plasma was investigated. The DPP IV activity in plasma samples obtained from health volunteers (n = 33) and diabetes patients (n = 36) was carefully measured by using GP-DM as a probe, as shown in Figure 3a. Notably, the plasma DPP IV activity level increased significantly in diabetes mellitus patients, with individual and mean values of DPP IV activity as high as 0.87 and 0.47 pmol/min/mg protein, respectively. The dramatic change in DPP IV activity demonstrates the potential of GP-DM as an effective probe for DPP IV in diagnosis, prognosis evaluation and even individual



tailoring of DPP IV inhibition-based therapy for diabetes mellitus. Additionally, DPP IV protein levels in human plasma samples were also determined by ELISA, which were consistent with the rates of GP-DM hydrolysis well (Figure 3b). These findings strongly suggest that GP-DM could serve as a useful sensor tool for trace DPP IV analysis in human plasma, and GP-DM based DPP IV activity determination was an effective diagnostic measure for diabetes. Subsequently, GP-DM was also applied to determine the activity of DPP IV in different human tissue preparations. The activities of DPP IV were extremely varied in liver, intestinal and kidney microsomes, of which HKM showing the highest DPP IV activity (Supplementary Figure S9). It demonstrated that GP-DM could sensitively measure the DPP IV activity in various human samples, implying the potential utility of GP-DM for DPP IV activity assessment, early detection of pathologies and individual tailoring of drug therapy.

Figure 3. a) Activity levels of endogenous DPP IV determined using GP-DM in the plasma of healthy volunteers and diabetes patients. b) Correlation analysis between the protein expression of DPP IV and reaction rates of GP-DM in individual human plasma. The DPP IV hydrolysis rates were measured after incubating the plasma with GP-DM for 30 min. The plasma DPP IV protein levels were also measured by ELISA. Fluorescence imaging of DPP IV in living cells. Considering the excellent sensing


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performance of GP-DM in DPP IV imaging, GP-DM was employed for the fluorescence imaging of cellular DPP IV (Figure 4). After incubation of LoVo and HepG2 cells with GP-DM at 37 °C for 60 min, strong fluorescence enhancement was observed in the LoVo and HepG2 cells. To validate the finding that this fluorescence turn-on specifically occurred due to the cellular DPP IV, chemical inhibition experiments were also performed, where LoVo cells were treated with sitagliptin (100 µM, selective inhibitor of GP-DM) for 30 min prior to the addition of GP-DM. As shown in Supplementary Figure S10, under the same imaging conditions, sitagliptin-pretreated LoVo cells exhibited a largely suppressed fluorescence signal; these findings all suggest that the obvious fluorescence enhancement in Figure 4c is mainly due to the DPP IV-mediated hydrolysis of GP-DM. We further confirmed the specificity of GP-DM for endogenous DPP IV in DPP IV knockdown LoVo cells. As expected, a dramatic change in the fluorescence signal was also observed (Supplementary Figure S11, S12). Furthermore, CCK-8 assay demonstrated that the viability of LoVo and HepG2 cells was greater than 90% when 25 µM GP-DM or 10 µM ADM was added for 12 h (Supplementary Figure S13, S14), demonstrating its good biocompatibility. The above results fully indicated that GP-DM had the good cell membrane-permeability for effectively sensing DPP IV in living cells.



Figure 4. Confocal laser scanning microscopy (CLSM) images of LoVo cells (a−d) and HepG2 cells (e−h). (a and e) Bright field images; (b and f) stained with 2 µM of Hoechst 33342; (c and g) stained with 10 µM GP-DM; (d) overlay image of (a), (b) and (c); (h) overlay image of (e), (f) and (g). The scale bar is 25 µm. Channel 1 for Hoechst 33342: excitation, 405 nm; emission, 430−490 nm. Channel 2 for GP-DM: excitation, 458 nm; semiconductor laser emission, 630−690 nm. Bioimaging of GP-DM in migration and proliferation assays. Migration and proliferation are the core hallmarks of cancer, which manifests with unlimited replicative potential and high clonogenic ability. To identify the effect of DPP IV on migration and proliferation, wound healing and colony formation assays were performed in LoVo cells and the corresponding DPP IV knockdown cells by using GP-DM for DPP IV bioimaging. These results observed in the wound-healing assay revealed the biological effect of DPP IV on tumor cell mobility in LoVo cells (Figure 5). As shown in Figure 5a and 5e, in the siRNA control group, the wound space between cell layers was almost occupied by the migrating cells after 48 h. In contrast, the cells treated with DPP IV siRNA failed to occupy the scraped space through migration, due to their impaired migration capability, with the migration rate decreasing from 78.3% to 3.2% (Figure 5c and 5g).


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Figure 5. Analysis of migration in LoVo cells (a, b, e, and f) and the corresponding DPP IV knockdown cells (c, d, g, and h) by fluorescence imaging of DPP IV. (a and c) Fluorescence images of DPP IV in LoVo cells and the corresponding DPP IV knockdown cells at 0 h; (b and d) overlay of the bright field image and the corresponding fluorescence image (a) and (c); (e and g) DPP IV in LoVo cells and the corresponding DPP IV knockdown cells at 48 h; (f and h) overlay of the bright field image and the corresponding fluorescence image (e) and (g); the scale bar is 250 µm. Stained with 10 µM of GP-DM for 30 min: excitation, 458 nm; semiconductor laser emission, 630−690 nm. Encouraged by the above-mentioned results for imaging the role of DPP IV in cell mobility, a clonogenic cell survival assay was employed to evaluate the effect of DPP IV on the clonogenic capacity of LoVo cells. Therefore, we further used the probe to monitor DPP IV, image the clonal formation of LoVo cells, and evaluate the change in colony formation under the different expression levels of DPP IV. In Figure 6, a remarkable decrease in the colony formation ratio and fluorescence intensity was observed in LoVo cells treated with DPP IV siRNA. Additionally, DPP IV knockdown significantly suppressed the proliferation by 72.3% relative to that of the controls; these findings all indicated that DPP IV can promote cell proliferation and attachment to neighboring cells. The present results demonstrate that GP-DM could be effectively used for DPP IV imaging in biological experiments, such as



migration and colony formation assays. Furthermore, the results implied that DPP IV played an important role in the acceleration of cancer progression, including proliferation and migration, while its inhibition may be a promising anti-cancer therapeutic approach.

Figure 6. Analysis of colony formation in LoVo cells (a and b) and the corresponding DPP IV knockdown cells (c and d) by fluorescence imaging of DPP IV. (a and c) Fluorescence images of DPP IV in LoVo cells and the corresponding DPP-IV knockdown cells; (b and d) overlay of the brightfield image and the corresponding fluorescence image (a) and (c); the scale bar is 750 µm. Stained with 10 µM of GP-DM for 30 min: excitation, 458 nm; semiconductor laser emission, 630−690 nm.

In vivo imaging of DPP IV in tumor-bearing mice. NIR light usually has some advantages, such as low background autofluorescence interference in animal tissue and minimal damage to biosamples; thus, many NIR fluorescent probes with excellent photophysical properties have been developed for in vivo imaging.32-36 Thus, the potential of GP-DM for imaging living animal models, including mouse and zebrafish, was evaluated. First, the suitability of GP-DM for tumor bioimaging in HepG2 and LoVo cell-bearing xenograft mice was investigated. After direct injection of GP-DM in physiological saline into the tumor of a living mouse, the accumulation of our probe was clearly visualized using in vivo whole-body fluorescence imaging with emission at 670-730 nm. After an intratumoral injection of GP-DM, the tumor region showed a significant enhancement in fluorescence intensity within 20 min, and the highest fluorescence intensity at the injection site was


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observed within 30 min, as shown in Figure 7a. With extension of the tracking time, the increased fluorescence intensity gradually extended from the injection site and diffused to the whole tumor region. As verified previously, after injecting GP-DM probe into the tumor, GP-DM was converted to ADM by the dysregulated DPP IV in tumors, causing fluorescence of the whole tumor that lasted more than 1 hour, which might be helpful for image-guided tumor resection. Inspired by these results, we continued to investigate the utility of GP-DM for imaging DPP IV in tumor tissue sections that were obtained from the xenograft tumor mouse model. The slices were subjected to GP-DM, immunohistochemical and hematoxylin-eosin (HE) staining, respectively. Our results strongly indicated that the remarkable fluorescence image induced by DPP IV activity is in good accordance with the immunohistochemical staining with DPP IV antibodies (Figure 7b). Taken together, these findings strongly suggest that GP-DM is a useful tool for imaging DPP IV in tumors and their tissue sections based on their distinctive DPP IV activity, which could be used for surgical diagnosis and therapy of cancers.



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Figure 7. a) In vivo NIR fluorescence imaging of DPP IV in tumor-bearing nude mice after tumor injection; b) Fluorescence and immunofluorescence bioimaging of the DPP IV in the tumor tissue slices. (i and ii) Fluorescence imaging of the DPP IV in the tumor tissue slices after incubation with GP-DM for 30 min and staining with 10 µM GP-DM for 30 min: excitation,

458

nm;

semiconductor

laser

emission,

630-690

nm.

(iii

and

iv)

Immunofluorescence images of the DPP IV in the tumor tissue slices. (v and vi) Hematoxylin-eosin staining of the tumor tissue slices. The scale bar is 50 µm. In vivo imaging of DPP IV in zebrafish. The zebrafish is a perfect animal model that shares a largely conserved physiology and anatomy with mammals and has thus widely been used in the fields of aquaculture, developmental biology, and even human health research.40-43 In particular, the unique features of zebrafish, such as the rapid development and transparency of their embryos, as well as the similarity of its organization and cell types to those of specific mammalian tissues (e.g. pancreas), facilitate both the probing of the role of DPP IV in the development of diabetes and tumors and the efforts to develop novel approaches to cure these increasingly widespread diseases.44-45 However, few studies have been reported that visualize DPP IV during the growth and development of zebrafish. On the basis of the excellent performance of GP-DM, herein we evaluated the in vivo tracking ability of GP-DM in zebrafish during their development periods. In our experiments, zebrafish grew in E3 embryo medium for different periods of time (1–7 days) and were then subjected to fluorescence imaging. We did not observe any fluorescence signal in the fishes as the control group, which was not treated with the probe, implying the imaging of DPP IV was GP-DM dependent (Supplementary Figure S15). As shown in Figure 8, zebrafish embryos at different stages of development display an obvious fluorescence, even in the early embryonic stage. This finding implies that zebrafish contain detectable endogenous DPP IV throughout the different


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embryonic development stages. Zebrafish embryos at 1 and 3 days had a clear and strong fluorescence region, precisely corresponding to the foregut of zebrafish, according to its anatomy.46,47 Subsequently, the fluorescence regions of zebrafish embryos and larvae were mainly distributed in the liver, pancreas, and gastrointestinal tract, all of which consistent with the expression spectrum of the DPP IV that is highly expressed in the liver, exocrine pancreas, and small intestine.48 Our present results accurately reflected the specific expression sites and functional activity of DPP IV throughout the life cycle of zebrafish for the first time. This finding suggests that GP-DM can be successfully used for in vivo sensing of DPP IV and for exploring the potential role of DPP IV in physiological and pathological changes.

Figure 8. Fluorescence images of DPP IV in living zebrafish grown for different periods (1–7 days). Fluorescence (left); brightfield (middle); merge (right). a) 1-day old; b) 3-day old; c)



5-day old; and d) 7-day old. Zebrafishes were treated with GP-DM (5 µM) for 60 min. CONCLUSION In conclusion, we synthesized and characterized an enzyme-activatable near-infrared fluorescent probe, GP-DM, for in vivo bioimaging of DPP IV. GP-DM emits significant turn-on NIR fluorescent signals in response to endogenous DPP IV activity, making it favorable for accurately and dynamically monitoring DPP IV activity in vitro and in vivo. The probe GP-DM exhibits excellent specificity and sensitivity in DPP IV imaging, as indicated by its higher catalytic activity than other human serine hydrolases and by its strong anti-interference ability with a complex biological matrix. We successfully used GP-DM to detect DPP IV activity in various biological samples and living cells and performed real-time in vivo imaging of DPP IV in zebrafish and tumor-bearing nude mice. These results all highlight the potential application value for early pathology detection, individual tailoring of drug therapy, and image-guided tumor resection. Furthermore, our results reveal that DPP IV, a key target enzyme, is closely associated with the migration and proliferation of cancer cells, and the regulation of DPP IV may be a promising anti-cancer therapeutic approach.

AUTHOR INFORMATION Corresponding Author

* E-mail: [email protected] & [email protected] Author Contributions

†These authors contributed equally (T. Liu and J. Ning) T. Liu and J. Ning contributed to the design of the research study, conducted experiments,


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acquired and analyzed data, and wrote the manuscript. B. Wang, B. Dong, S. Li, X.G. Tian, Z.L. Yu and Y.L. Peng conducted experiments and aided in the acquisition of data. C. Wang, X.Y. Zhao, X.K. Huo and C.P. Sun aided in data acquisition and analyses. J.N. Cui contributed to the design of the research study. L. Feng and X.C. Ma contributed to the design of the research study and helped write the manuscript.

ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (No. 81622047, 81503201, 81473334 and 21572029), the Dalian Outstanding Youth Science and Technology Talent program (2015J12JH201) and the Distinguished Professor of Liaoning Province program for financial support, as well as the State Key Laboratory of Fine Chemicals (KF1603).

Supporting Information Details regarding the synthesis and structure characterization of the probe GP-DM, the specificity, kinetics studies, representative fluorescence images, Western blots results, and the cell toxicity of GP-DM and ADM are provided in the supplementary material.

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An activatable near-infrared fluorescent probe for dipeptidyl peptidase

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