Near-Infrared Fluorogenic Probes with Polarity-Sensitive Emission for

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Near-Infrared Fluorogenic Probes with Polarity-Sensitive Emission for in Vivo Imaging of an Ovarian Cancer Biomarker Defan Yao,† Zhi Lin,‡ and Junchen Wu*,† †

Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China ‡ College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China S Supporting Information *

ABSTRACT: Lysophosphatidic acid (LPA, cutoff values ≥ 1.5 μM) is an effective biomarker for early stage ovarian cancer. The development of selective probes for LPA detection is therefore critical for early clinical diagnosis. Although current methods have been developed for the detection of LPA in solution, they cannot be used for tracking LPA in vivo. Here, we report a near-infrared (NIR) fluorescent probe that can selectively respond to LPA based on polarity-sensitive emission at a very low detection limit of 0.5 μM in situ. This probe exhibits a marked increase of fluorescence at 720 nm upon binding to LPA, allowing the direct visualization of LPA in vitro and in vivo without interference from other biomolecules. Moreover, the probe containing two arginine-glycine-aspartic acid units can be efficiently taken up by cancer cells based on an αvβ3 integrin receptor targeting mechanism. It also exhibits excellent biocompatibility and high pH stability in live cells and in vivo. Confocal laser scanning microscopy and flow cytometric imaging of SKOV-3 cells have confirmed that our probe can be used to image LPA in live cells. In particular, its NIR turn-on fluorescence can be used to effectively monitor LPA imaging in a SKOV-3 tumor-bearing mouse model. Our probe may pave the way for the detection of cancer-related biomarkers and even for early stage cancer diagnosis. KEYWORDS: lysophosphatidic acid, biomarker, polarity-sensitive, near-infrared, probe

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Very recently, several fluorescence probes have been extended to biosensing and imaging applications, including fluorogenic squaraine dimers for ligand-protein affinity labeling,22 self-assembled squaraine dye nanoparticles for selective detection of human serum albumin protein in clinical serum diagnosis,23 and tetraphenylsilole integrated with a cyclic arginine-glycine-aspartic acid (cRGD) peptide for detection and imaging of αvβ3 integrin receptors.24 Despite these advances, the selective detection of LPA using these kinds of probes remains challenging. Near-infrared (NIR) fluorescence probes are particularly promising for biological applications within a desirable optical window (900 nm > λem > 650 nm) because they can minimize background autofluorescence interference from cancer cells, serum, and tissues.25−35 Given the absorption and emission bands of squaraine dyes in the NIR region, we sought to develop a turn-on probe that responds to physiological levels of LPA in vitro and in vivo via polarity-sensitive changes in squaraine dyes for application in the early clinical diagnosis of ovarian cancer.

INTRODUCTION Molecular probes for the selective sensing and detection of tumor-specific biomarkers have attracted considerable research interest due to their potential applications in early clinical diagnosis.1−4 Lysophosphatidic acid (LPA), a lipid mediator that functions as a potent inducer of cell proliferation, migration, and survival, is widely distributed in cancerous cells and tissues.5,6 It is an ideal biomarker for ovarian cancer at cutoff values ≥ 1.5 μM, showing 100 and 90% sensitivity for advanced disease and early disease, respectively, and a specificity of 90%.7−9 Current LPA detection methods, including enzymatic cycling,10 surface-enhanced Raman scattering (SERS) using silver nanoparticles,11 polyclonal antibody immunoassay,12 lanthanide zeolite-like metal−organic frameworks (Ln-ZMOFs),13 liquid chromatograph−mass spectrometry (LC-MS),14 and chemical probes15−17 enhance the LPA signals of only limited amounts in vitro. Thus, the application of currently available sensors to detect LPA in live cells and in vivo is largely limited by their low sensitivity. Nevertheless, as was reported by Mills and Moolenaar,18,19 LPA contributes to polarity changes in the tumor microenvironment that can promote cancer progression. Therefore, the development of specific probes for LPA detection could provide a key to improving the early clinical diagnosis of ovarian cancer.20,21 © XXXX American Chemical Society

Received: December 4, 2015 Accepted: February 18, 2016

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DOI: 10.1021/acsami.5b11826 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Structures of LPA, RGDSq2, and G4RGDSq2

undergoes a conformational change from a folded state to an extended form that breaks the hydrogen bonds between guanines,38 and the squaraine units correnspondingly recover the NIR fluorescence. RGD peptides incoporated in probe G4RGDSq2 enable the overall molecule to have adequate polarity for applications in water and also the capability to be efficiently taken up by cells based on an αvβ3 integrin receptor targeting mechanism. 39,40 Thus, the designed probe G4RGDSq2 can be used to directly image the high levels of

Here, we designed an NIR probe for the selective imaging of LPA in live cells and in vivo (Scheme 1). The probe is a folded molecule consisting of two arginine-glycine-aspartic acid (RGD) sequences, four guanines, and two squaraine dyes. The fluorescence of probe G4RGDSq2, which is based on polarity-sensitive squaraine dyes, is largely quenched in aqueous solution by the conformational restriction that is enhanced by the formation of intramolecular hydrogen bonds between the four guanines.22,36,37 Upon binding to LPA, probe G4RGDSq2 B


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ACS Applied Materials & Interfaces

harvested cells were then suspended in PBS (pH 7.4). Each cell was quickly analyzed by an ImageStreamX Mark II flow cytometer. Channel 7 for DAPI: excitation, 405 nm; emission collected, 430−505 nm. Channel 11 for G4RGDSq2: excitation, 642 nm; semiconductor laser emission collected, 652−700 nm. Cytotoxicity Assay. The cytotoxicity was measured using a standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay with IOSE80 and SKOV-3 cells. Cells growing in log phase were seeded into 96-well cell-culture plates at 1 × 104 cells/well. The cells were incubated overnight at 37 °C under humidified conditions of 95% air and 5% CO2. G4RGDSq2 (100.0 μL/well) at concentrations of 2, 4, 6, 8, 10, and 20 μM in DMEM, and McCoy’s 5A was added to the wells of the treatment group, whereas for the final negative control group, 100.0 μL of DMEM and McCoy’s 5A were added. The cells were incubated for 24 h at 37 °C under humidified conditions of 95% air and 5% CO2. Subsequently, 10 μL of MTT solution (5 mg/mL in PBS) was added to the media for an additional 4 h incubation at 37 °C, allowing viable cells to reduce the yellow tetrazolium salt (MTT) into dark blue formazan, which precipitated as crystalline solids. After removal of the media, formazan extraction was performed with 100 μL of DMSO and its quantity was determined colorimetrically using a Multimode Plate Reader (BioTek, USA) at 490 nm (absorbance value). The following formula was used to calculate the viability of cell growth: viability (%) = mean absorbance value of the treatment group-blank/mean absorbance value of the control-blank × 100. Confocal Microscopic Imaging for Cells. SKOV-3 and IOSE80 cells were seeded in a 35 mm Petri dish with a glass cover slide and allowed to adhere overnight before treatment. The cells were incubated with DAPI (1 μg/mL) in McCoy’s 5A and DMEM, respectively, for 8 h at 37 °C under humidified conditions of 95% air and 5% CO2. After washing the cells with PBS (pH 7.4, 1 mL × 3 times), the SKOV-3 and IOSE80 cells were incubated with G4RGDSq2 (10 μM) in McCoy’s 5A and DMEM for 3 h at 37 °C under humidified conditions of 95% air and 5% CO2, respectively. Cell imaging was then carried out after washing the cells with PBS (pH 7.4, 1 mL × 3 times). Cell fluorescence images were obtained with a confocal laser scanning microscope (Nikon A1, Japan, 60× oilimmersion objective lens). Channel 1 for DAPI: excitation, 405 nm; emission collected, 425−475 nm. Channel 2 for G4RGDSq2: excitation, 638 nm; emission collected, 664−735 nm. Murine Tumor Model. All animal handling was performed in accordance with Animal Research Committee guidelines of East China University of Science and Technology and conformed to the guide for the care and use of laboratory animals. SKOV3 cells were washed with PBS (pH 7.4) and harvested using 0.05% Trypsin/EDTA (Sigma). After centrifugation, the harvested cells were then suspended in PBS (pH 7.4). Six-week-old (approximately 20 g) female BALB/c nude mice (Shanghai Slac Laboratory Animal Co. Ltd., China) were implanted subcutaneously on the right flank with 10 million cells of SKOV-3 in 0.2 mL of PBS (pH 7.4), and tumors developed within 4 weeks. Mice were used for fluorescence in vivo imaging when the tumors were 8−10 mm in diameter. In Vivo Fluorescence Imaging. The mice were imaged using an IVIS spectrum series in vivo imaging system (PerkinElmer). Mice were anesthetized by 2% isoflurane inhalation for 30 s in an anaerobic box before fluorescence in vivo imaging each time. After mice were injected intravenously with G4RGDSq2 (20 nmol) in 0.1 mL of PBS (pH 7.4), they were placed in the IVIS spectrum series in vivo imaging system (PerkinElmer) and scanned to determine the endogenous signal of tumors (excitation: 710 nm, emission collected: 740−760 nm).

LPA in the intracellular milieu based on the activatable emission in situ.

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EXPERIMENTAL SECTION

Materials and Reagents. Unless otherwise stated, all organic solvents were dried and distilled before use. Water was purified by a Millipore filtration system. All reagents and chemicals were AR grade and used without further purification unless otherwise noted. Reactions were performed under an argon atmosphere unless otherwise specified. Analytical thin-layer chromatography (TLC) was performed on silica gel plates using UV light as a visualizing agent. Calf thymus DNA (ctDNA), nicotinamide adenine dinucleotide (NAD+), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), adenosine triphosphate (ATP), albumin from bovine serum (BSA), glutathione (GSH), LPA, and S1P were purchased from Sigma. Fluorescence and UV−Vis Experiments. Fluorescence spectra were measured with a Fluoromax-4 Spectrofluorometer (HORIBA Scientific), and absorption spectra were recorded using a Cary 100 UV−visible spectrophotometer (Agilent Technologies) at 25 °C. The slit widths were set to 5 nm for excitation and emission. The data points were collected at 1.0 nm increments with a 0.1 s integration time. All spectra were corrected for intensity taking advantage of the manufacturer-supplied correction factors, and they were corrected for background fluorescence and absorption by subtracting a blank scan of the buffer. RGDSq2, G4RGDSq2, and LPA were all dissolved in TBS (pH 7.4, 50 mM Tris, 25 °C). CD Spectroscopy. The CD spectra of RGDSq2 and G4RGDSq2 (10 μM) were recorded on a JASCO J-815 CD spectrometer at 25 °C. The spectra were scanned in a quartz optical cell of 1.0 cm path length, and the samples were dissolved in TBS (pH 7.4, 50 mM Tris, 25 °C). Data were collected at 1.0 nm increments with a 0.1 s integration time from 350 to 190 nm at each wavelength. ELISA for the Determination of Phospholipids. The amounts of phospholipids in IOSE80 and SKOV-3 cells, including LPA, LPC, S1P, PA, and PS, were measured using the ABC-double antibody sandwich ELISA method. LPA, S1P, PA, PS, and LPC kits (human, double antibody method, and 96-well) were purchased from Jonln, Ltd. (Shanghai). The intracellular components of IOSE80 and SKOV3 cells (the concentration of cells is 1 × 106 cells/mL) were prepared based on the manufacturer’s instructions and directly used in the ELISA assay. The ELISA assay experiments were performed according to literature procedures.51,52 Cell Culture. IOSE80 (immortalized ovarian surface epithelium) and SKOV-3 (human ovarian carcinoma epithelial) cells were purchased from Shanghai Bogoo Biotech Co., Ltd., China. IOSE80 cells were cultured in DMEM (Gibco) at 37 °C under humidified conditions of 95% air and 5% CO2. SKOV-3 cells were cultured in McCoy’s 5A (Gibco) at 37 °C under humidified conditions of 95% air and 5% CO2. All media were supplemented with 10% fetal bovine serum, 100 U penicillin, and 0.1 mg of streptomycin (Gibco) per milliliter. The culture media were changed every 2 days to maintain exponential growth of the cells. Cells were passaged using 0.05% Trypsin/EDTA (Sigma) when they reached 80−90% confluence and seeded for the experiments. Flow Cytometry. The cell uptake of G4RGDSq2 was measured by an ImageStreamX Mark II imaging flow cytometer. IOSE80 and SKOV-3 cells were seeded into 6-well plates (1.0 mL cell suspension per well) at a density of 1 × 106 cells/mL in DMEM and McCoy’s 5A, respectively, and allowed to adhere overnight before treatment. IOSE80 and SKOV-3 cells were incubated with DAPI (1.0 μg/mL) for 8 h at 37 °C under humidified conditions of 95% air and 5% CO2. Cells incubated with DAPI (1.0 μg/mL) in DMEM and McCoy’s 5A were set as the control. After washing with PBS (pH 7.4), the cells were treated with G4RGDSq2 at concentrations of 0.5−2.0 μM in DMEM and McCoy’s 5A, respectively, and incubated for 3 h at 37 °C. The media were then removed, and the cells were washed three times with cold PBS (pH 7.4). The cells were harvested using 0.05% Trypsin/EDTA (Sigma) and centrifuged at 1000 rpm for 5 min. The

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RESULTS AND DISCUSSION The unsymmetrical squaraine dyes and Fmoc-protected guanines with an N-(2-aminoethyl)-glycine backbone were first synthesized according to literature reports (Schemes S1 and S2).41−43 Thereafter, probe G4RGDSq2 was synthesized using a standard Fmoc-based solid-phase peptide (SPPS) procedure. This probe was synthesized on a rink amide resin C


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Figure 1. (a) Absorption and (b) fluorescence spectra (λex = 700 nm) of 1.0 μM G4RGDSq2 in TBS-DMSO solution. The DMSO content (%) by volume was 100/0, 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 50/50, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/ 10, 95/5, and 0/100. (c) Absorption and (d) fluorescence spectrum changes for the 1.0 μM G4RGDSq2 (λex = 700 nm) upon the addition of LPA (0−10 equiv) in TBS (50 mM Tris, pH 7.4, 25 °C).

Tris, pH 7.4, 25 °C) (Figure S3a, b). Two absorption bands at approximately 650 and 710 nm were observed for RGDSq2 and G4RGDSq2. The emission of G4RGDSq2 at 720 nm was heavily quenched compared with that of RGDSq2. Probe G4RGDSq2 had near-zero fluorescence background signals, which make it a promising candidate for subsequent studies. Next, we examined the sensing abilities of G4RGDSq2 in solvents of various polarities based on the absorption and fluorescence spectra. G4RGDSq2 (1 μM) was dissolved in dimethyl sulfoxide (DMSO) and TBS. When the DMSO content increased, the absorption bands of G4RGDSq2 at 655 and 710 nm also gradually increased (Figure 1a), and the fluorescence of G4RGDSq2 concomitantly increased and redshifted to 725 nm (Figure 1b). The absolute quantum yield (Φ) of G4RGDSq2 reached up to 6.0% in DMSO, which is higher than that in TBS alone (0.37%) (Table S1).44 The absorption and fluorescence properties of G4RGDSq2 in various concentrations of dimethylformamide (DMF) were further studied. With increasing DMF content (up to 80%), the absorption bands of G4RGDSq2 at 650 and 705 nm gradually increased. In addition, the emissions of G4RGDSq2 red-shifted from 710 to 720 nm at a quantum yield of 2.88% (Figure S4 and Table S1). Considering these observations, the binding of G4RGDSq2 to LPA in TBS was also examined using fluorescence and absorption spectroscopy. In the absence of LPA, we observed marked absorption bands at 650 and 710 nm (Figure 1c). Upon the titration of increasing amounts of LPA (0−10 equiv), the emission of G4RGDSq2 at 720 nm was enhanced from a

(loading: 0.81 mmol/g) by a CEM Liberty one microwaveassisted peptide synthesizer. Each amino acid coupling was carried out under microwave radio conditions (35 W, 60 ± 5 °C, 20 min). The Fmoc-protected group was removed with 20% piperidine in DMF (2 × 20 mL, 5 min each) under microwave radio conditions (35 W, 60 ± 5 °C, 5 min). The resin was washed with DMF (7 × 10 mL, ∼1 min each) to remove the last traces of the amino acid and piperidine. When all of the amino acids were attached, the products were transferred into a glass peptide synthesis vessel. After the Fmoc was deprotected, the products were coupled with two Fmocprotected guanine monomers. Followed by Fmoc deprotection, the products were further reacted with unsymmetrical squaraine dyes. On the basis of the similar procedure described, RGDSq2, which was used as a control, was also synthesized without the four guanine nucleobases (Scheme 1). After completion of RGDSq2 and G4RGDSq2 synthesis, the final products were cleaved from their solid support by treatment with a cleavage cocktail of trifluoroacetic acidtriisopropylsilane-H2O (95:2.5:2.5) for 2 h. After removing the solution, the deep green solids were precipitated with dry diethyl ether and centrifuged at 6500 rpm for ∼15 min. The crude products were purified and isolated by preparative high performance liquid chromatography (HPLC) on a C-18 column. They were further characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Schemes S3 and S4 and Figures S1 and S2). We first tested the absorption and emission spectra of G4RGDSq2 and control RGDSq2 in Tris buffer (TBS, 50 mM D


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Figure 2. (a) Fluorescence response of G4RGDSq2 (10 μM) at 720 nm (λex 700 nm) upon the addition of various species (40 μM) in TBS (50 mM Tris, pH 7.4, 25 °C); Inset is the fluorescence spectra of G4RGDSq2 bound to various biomolecules. (b) Fluorescence intensity of G4RGDSq2 (10 μM) at 720 nm (λex 700 nm): red columns represent the fluorescence of G4RGDSq2 in the presence of various species (4.0 equiv), and blue columns represent G4RGDSq2 with various species (4.0 equiv) and LPA (4.0 equiv) in TBS (50 mM Tris, pH 7.4, 25 °C). (c) Normal mouse blood serum as a control and (d) SKOV-3 tumor-bearing mouse blood serum. Fluorescence intensity changes for G4RGDSq2 (10 μM) at 720 nm (λex 700 nm) upon the addition of serum aliquots (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 μL) in TBS (50 mM Tris, pH 7.4, 25 °C).

of LPA at the same pH conditions (zeta potential: −24.60 ± 2.80 mV) (Figure S9). The results suggest that the electrostatic interactions are the main binding force between G4RGDSq2 and LPA. The LPA binding-sites of G4RGDSq2 were also confirmed by fluorescence and circular dichroism (CD) spectroscopy studies. Upon binding to LPA (4.0 equiv), the fluorescence of G4RGDSq2 and RGDSq2 at 720 nm exhibited almost identical intensities (Figure S3c, d). Both RGDSq2 and G4RGDSq2 contain the β-turn structure, which produced a characteristic CD spectra consisting of a strong, positive band at ∼205 nm and a weak, negative band at ∼224 nm (Figure S10).45,46 G4RGDSq2 also showed two additional positive CD bands at approximately 245 and 280 nm that are characteristic of the interaction between guanines.47−49 Upon the addition of LPA (4.0 equiv), the CD bands of G4RGDSq2 at approximately 245 and 280 nm decreased significantly and nearly vanished. The CD bands of G4RGDSq2 corresponding to the β-turn motif changed markedly, which is consistent with the behavior of RGDSq2. In conclusion, our results demonstrate that G4RGDSq2 binds to LPA predominantly through electrostatic interactions based on the recognition units of RGD rather than guanines. Competitive fluorescence assays were performed to evaluate the selectivity of G4RGDSq2 in LPA detection. As shown in

quantum yield of 0.37% to a quantum yield of 6.0% (Figure 1d and Table S1). Interestingly, the spectral shape changes of Figure 1c were consistent with that of Figure 1a in the DMSO (0−30%) content, which indicated the polar contribution of LPA (0−10 equiv) was nearly equal to that of the DMSO (0− 30%) content in TBS. The stability of G4RGDSq2 in the presence and absence of LPA (4.0 equiv) at various pH values (pH 4.0, 7.4, and 8.5) was also assessed using absorption and fluorescence spectroscopy. It was determined that pH values made no significant difference to the fluorescence emission and absorption peaks of G4RGDSq2 (Figure S5). The interactions between G4RGDSq2 and LPA were further studied by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zeta potential analysis. In the DLS experiments, G4RGDSq2 and free LPA formed different-sized particles with diameters of 126 and 385 nm in TBS, respectively. Upon the addition of LPA (4.0 equiv) to a TBS solution of G4RGDSq2, the size of the G4RGDSq2−LPA complex increased to 585 nm (Figure S7). This observation is consistent with the TEM results (Figure S8) that also indicate binding of LPA to G4RGDSq2. The zeta potential measurements showed that LPA is negatively charged (zeta potential: −37.63 ± 1.27 mV) and that G4RGDSq2 is positively charged (zeta potential: 11.23 ± 1.77 mV) in TBS. The zeta potential of the G4RGDSq2−LPA complex is significantly higher than that E


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ACS Applied Materials & Interfaces Figure 2a, the fluorescence intensity of G4RGDSq2 bound to LPA (4.0 equiv) at 720 nm increased by up to 20-fold compared to that of G4RGDSq2 alone. The addition of equivalent amounts (4.0 equiv) of other biological species, such as nucleotides, amino acids, and calf thymus DNA (ctDNA), caused no significant increase in the fluorescence. The only exceptions were bovine serum albumin (BSA), a structurally similar phospholipid, and expressed sphingosine 1-phosphate (S1P) in ovarian cells,50 which also resulted in an increase in fluorescence, albeit to a significantly lower extent (only 30%) than that of LPA. In the titration experiments, the lower detection limit of LPA by G4RGDSq2 was estimated to be 0.5 μM (Figure S11). Moreover, the results from the competitive fluorescence assay indicate that most of the tested species do not bind appreciably to G4RGDSq2 (Figure 2b). The practical utility of the probe in blood serum was further investigated using fluorescence spectroscopy. Blood serum samples were obtained from two normal and xenograft tumor model mice. The normal mouse blood serum was used as a control. Upon the titration of 5 μL of serum into a TBS solution of G4RGDSq2 (10 μM), the fluorescence intensity of the xenograft tumor blood serum sample was increased by approximately 2.5-fold compared with that of the normal mouse blood serum (Figure 2c, d). Futhermore, in agreement with Figure 2d, the fluorescent signals of probe G4RGDSq2 (Figure S6) were enhanced upon the titration of 5.0 μL of normal mouse blood serum with the LPA content (40 μM) into a TBS solution of G4RGDSq2 (10 μM). Thus, probe G4RGDSq2 can be used to detect LPA in complex biological media. For determining the levels of phospholipids in human normal ovarian surface epithelial cells (IOSE80) and human ovarian adenocarcinoma cells (SKOV-3), the concentration of common intracellular phospholipids, such as LPA, S1P, lysophosphatidylcholine (LPC), phosphatidic acid (PA), and phosphatidylserine (PS) were measured using an enzymelinked immunosorbent assay (ELISA).51,52 As shown in Figure 3 and Table S2, LPA was expressed at a high level in SKOV-3

cytoplasmic phospholipids tested were in line with previously reported results.53−56 Thus, we could deduce that the observed polarity changes in the SKOV-3 cells were largely due to the presence of LPA. We next investigated whether G4RGDSq2 could be used as a turn-on probe to image high levels of LPA in SKOV-3 cells in situ and whether G4RGDSq2 could be efficiently internalized into cells through ligand−receptor interactions. The cellular uptake of G4RGDSq2 in SKOV-3 cells was quantitatively evaluated using flow cytometry (FCM).57,58 IOSE80 cells were used as a control. Figure 4 and Figure S12 indicate the

Figure 4. MFI values of IOSE80 (gray) and SKOV-3 (green) cells treated with different concentrations of G4RGDSq2 (0.5−2.0 μM) in DMEM and McCoy’s 5A media, respectively.

significant differences in fluorescence intensity between SKOV3 and IOSE80 cells after incubation with G4RGDSq2 at concentrations of 0.5−2.0 μM in DMEM and McCoy’s 5A media, respectively. The mean fluorescence intensity (MFI) values of SKOV-3 cells, compared with that of the IOSE80 cells, increased approximately 3.0−6.0-fold in the concentration range from 0.5 to 2.0 μM (Figure 4). Confocal laser scanning microscopy (CLSM) of SKOV-3 and IOSE80 cells was performed after incubation with G4RGDSq2 and DAPI, which is a nuclear stain with a blue signal (Figure 5b, d, f, h). A considerably weaker red fluorescence signal was observed in the IOSE80 cells (Figure 5c). In contrast, significantly stronger red fluorescence signals were observed in the SKOV-3 cells (Figure 5g). The overlay images revealed the presence of G4RGDSq2 in the cytoplasm of the IOSE80 and SKOV-3 cells (Figure 5d, h), which was consistent with the FCM images (Figure S13).59 These results imply that fluorescence signals from G4RGDSq2 were most likely a result of the polarity changes in the SKOV-3 cells from the high levels of LPA. For further confirming that G4RGDSq2 was internalized into cells through ligand−receptor interactions and that the red emission of G4RGDSq2 was generated from the high levels of LPA, SKOV-3 and IOSE80 cells were cultured with an effective αvβ3 integrin inhibitor (cilengitide, 4.0 μM)60 and an LPA inhibitor (S32826, 5.0 μM)61 prior to the administration of G4RGDSq2 (10 μM). Much lower red fluorescence signals were observed in SKOV-3 and IOSE80 cells from confocal laser scanning microscopy (CLSM) images (Figures S14 and S15). These results are consistent with G4RGDSq2 being internalized into cells through ligand−receptor interactions and binding LPA at high LPA levels, which generated a red

Figure 3. Quantification of phospholipids, including LPA, S1P, PA, PS, and LPC, for IOSE80 and SKOV-3 cells by ELISA.

cells (289 nmol/L) and at a relatively low level in IOSE80 cells (170 nmol/L). S1P was expressed in both SKOV-3 cells (100 nmol/L) and IOSE80 cells (85 nmol/L), but was generally at reduced levels. The other phospholipids were present at considerably lower levels in the intracellular milieu. For example, the levels of LPC reached only 0.33 and 0.31 nmol/ L in the SKOV-3 and IOSE80 cells, respectively. The levels of F


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Figure 5. CLSM images of IOSE80 cells (a−d) and SKOV-3 cells (e−h), respectively. (a and e) Bright-field images; (b and f) stained with 1 μg/mL of DAPI; (c and g) stained with 10 μM G4RGDSq2; (d) overlay image of (b) and (c); (h) overlay image of (f) and (g). Scale bar is 20 μm. Channel 1 for DAPI: excitation, 405 nm; emission collected, 425−475 nm. Channel 2 for G4RGDSq2: excitation, 638 nm; semiconductor laser emission collected, 664−735 nm.

fluorescence signal. This result also explains the observed changes in the FCM images when the cells were not treated with inhibitors (Figure S13). In addition, the cytotoxicity of G4RGDSq2 was measured using the MTT assay. The cell viabilities of IOSE80 and SKOV-3 cells were greater than 90% after 24 h incubation with 0−20 μM G4RGDSq2, demonstrating the low cytotoxicity of the probe (Figure 6).

xenograft mouse was intravenously injected without an inhibitor before the injection of G4RGDSq2. After 24 h, the four mice were euthanized by excess isoflurane inhalation, and their organs were dissected. Next, ex vivo NIR fluorescence imaging of G4RGDSq2 and quantitative fluorescence intensity measurements in the isolated organs and tumors were performed and analyzed using the IVIS spectrum small-animal in vivo imaging system (Figure 7a, b). For the two inhibitorinjected mice and the normal mouse, G4RGDSq2 mainly accumulated in the liver, spleen, and lungs with much lower accumulation in the stomach, kidneys, heart, and tumor (Figure 7a and Figure S16). In sharp contrast, without prior administration of the inhibitor, large amounts of G4RGDSq2 were observed in the tumor. Moreover, in vivo NIR fluorescence imaging of the G4RGDSq2-treated xenograft tumor mouse at 720 nm for 0−24 h revealed the gradual accumulation of the probe in the tumor compared with that of the normal and two inhibitor-treated mice (Figure S16). Threedimensional NIR fluorescence images confirmed that the signal originated from the tumor region (Figure 7c, d), which is consistent with the fluorescent imaging results of the isolated tumor (Figure 7a, b). The in vivo toxicity of G4RGDSq2 was further evaluated using optical microscopy based on histological changes observed in the normal organs. These results demonstrated that G4RGDSq2 does not cause significantly detrimental effects during imaging in live animals (Figure S17). Thus, G4RGDSq2 can be effectively introduced into SKOV-3 cells by binding to the αvβ3 integrin receptor, and it retains its activity in the presence of high levels of LPA in the tumor.

Figure 6. Cell viability values (%) estimated by MTT proliferation tests at different concentrations of G4RGDSq2. IOSE80 cells (gray) and SKOV-3 cells (green) were cultured in the presence of G4RGDSq2 (0−20 μM) at 37 °C for 24 h.

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To identify the distribution of LPA in the tumor and to determine the specificity of G4RGDSq2 in vivo, we intravenously injected G4RGDSq2 (20 nmol) into a normal mouse and three xenograft tumor mice with SKOV-3 cells. The normal mouse served as a control. Two xenograft mice were intravenously injected with an effective αvβ3 integrin inhibitor (cilengitide, 170 nmol) and an LPA inhibitor (S32826, 486 nmol) prior to the administration of G4RGDSq2.62,63 Another

CONCLUSIONS In conclusion, we have developed an effective fluorogenic probe, G4RGDSq2, based on polarity-sensitive NIR emission in situ. This probe exhibits high sensitivity and specificity for LPA in vitro and in vivo based on the specific recognition between the RGD ligand and αvβ3 integrin receptor. Moreover, the probe displays high pH stability in aqueous media and low G


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Figure 7. (a) Ex vivo NIR fluorescence imaging of G4RGDSq2 (20 nmol) distribution in isolated organs and tumors after intravenous injection. (b) Quantitative analysis of relative organ accumulation in the xenograft tumor model system mice and normal mouse. The error bars represent ± SD in the analysis of relative counts using the IVIS spectrum small-animal in vivo imaging system. (c) Three-dimensional NIR image reconstruction of the xenograft tumor mouse without inhibitor treatment using an IVIS spectrum series in vivo imaging system. (d) In vivo NIR fluorescence imaging of one normal mouse and one xenograft tumor-bearing mouse intravenously injected with G4RGDSq2 over 0−24 h (black circles indicate the tumor site). All images were acquired under the same instrument conditions.

for the Central Universities (WJ1213007) for financial support. We also thank Prof. He Tian for his constructive suggestions.

toxicity in live cells and in vivo. The turn-on NIR emission can be used to visualize LPA in a mouse xenograft tumor model system. The detection limit of 0.5 μM of G4RGDSq2 encourages potential applications for sensing LPA in serum and for the early clinical diagnosis of ovarian cancer. We believe that this probe can become an important detection tool for LPA imaging in the NIR optical window, and it could be further applied in a wide range of clinical diagnostics.

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(1) Mills, G. B.; Moolenaar, W. H. The Emerging Role of Lysophosphatidic Acid in Cancer. Nat. Rev. Cancer 2003, 3, 582−591. (2) Bast, R. C.; Hennessy, B.; Mills, G. B. The Biology of Ovarian Cancer: New Opportunities for Translation. Nat. Rev. Cancer 2009, 9, 415−428. (3) Zhang, H.; Fan, J.; Wang, J.; Zhang, S.; Dou, B.; Peng, X. An Off−On COX-2-Specific Fluorescent Probe: Targeting the Golgi Apparatus of Cancer Cells. J. Am. Chem. Soc. 2013, 135, 11663−11669. (4) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J. Cyanine-Based Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures and Live Mouse Tissues. J. Am. Chem. Soc. 2014, 136, 5351−5358. (5) Houben, A. J. S.; Moolenaar, W. H. Autotaxin and LPA Receptor Signaling in Cancer. Cancer Metastasis Rev. 2011, 30, 557−565. (6) Moolenaar, W. H. Lysophosphatidic Acid Signaling. Curr. Opin. Cell Biol. 1995, 7, 203−210. (7) Nossov, V.; Amneus, M.; Su, F.; Lang, J.; Janco, J. M. T.; Reddy, S. T.; Farias-Eisner, R. The Early Detection of Ovarian Cancer: From Traditional Methods to Proteomics. Can We Really Do Better than Serum CA-125? Am. J. Obstet. Gynecol. 2008, 199, 215−223. (8) Sutphen, R.; Xu, Y.; Wilbanks, G. D.; Fiorica, J.; Grendys, E. C.; LaPolla, J. P.; Arango, H.; Hoffman, M. S.; Martino, M.; Wakeley, K.; Griffin, D.; Blanco, R. W.; Cantor, A. B.; Xiao, Y.; Krischer, J. P. Lysophospholipids Are Potential Biomarkers of Ovarian Cancer. Cancer. Epidemiol. Biomarkers Prev. 2004, 13, 1185−1191. (9) Blaho, V. A.; Hla, T. Regulation of Mammalian Physiology, Development, and Disease by the Sphingosine 1-Phosphate and Lysophosphatidic Acid Receptors. Chem. Rev. 2011, 111, 6299−6320. (10) Kishimoto, T.; Matsuoka, T.; Imamura, S.; Mizuno, K. A Novel Colorimetric Assay for the Determination of Lysophosphatidic Acid in

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b11826. Synthesis of compounds, ESI-MS data, 1H NMR, 13C NMR, and UV−vis spectra, analysis of dynamic light scattering (DLS), TEM images, zeta potentials, CD spectra, ELISA experiments, cell experiments, and in vivo experiments (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Basic Research 973 Program (2013CB733700), NSFC/China (91529101 and 21572057), the Program of Introducing Talents of Discipline to Universities (B16017), and the Fundamental Research Funds H


Research Article

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