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1.IntroductionSince its discovery in 1993, an increasing amount of effort has been invested in studying the pharmacology and physiopathological role of type 2 cannabinoid receptors (). Presently, both academia and the pharmaceutical industries consider this receptor as a major therapeutic target. Specifically, represents a promising target to treat inflammation,1,2 pain,3 osteoporosis,4 autoimmune diseases,5 addiction,6,7 psychiatric disorders,8–10 diabetes,11–13 cardiovascular disorders,14 cancers, and neurodegenerative diseases. For example, recent studies have shown that plays a fundamental role in tumorigenesis and that the agonists may represent promising venues to develop cancer treatments.15,16 Specifically, some of the most exciting recent research carried out by several laboratories demonstrated that agonists potently inhibited viability, proliferation, adhesion, and migration of various cancer cells, such as breast,17–19 prostate,20,21 glioma,22 colon,23 lung,24 thyroid,25 lymphoma,26 skin,27 pancreas,28 and liver29 cancers. These data were collected from both cellular systems and preclinical animal models. Additional evidence indicates that the agonists exhibit promising therapeutic value for treating neurodegenerative diseases. agonists attenuate Alzheimer’s disease pathogenesis by blocking -amyloid peptide-induced activation of microglial cells30 and play a neuroprotective role in Huntington’s disease and amyotrophic lateral sclerosis.31 The richness of ’s regulatory roles has rendered this receptor as an attractive target to study a variety of diseases and biological processes. However, the precise role of in the regulation of diseases remains unclear. The ability to specifically image would contribute to develop reliable -based therapeutic approaches with a better understanding of the mechanism of action in these diseases. Little has been done to target for imaging studies and therapeutic evaluations. The current imaging techniques to identify rely heavily on immunostaining; however, all currently available antibodies have significant nonspecific binding issues, leading to unreliable imaging results.6,32 As such, the development of reliable contrast agents for imaging is critically needed in the field. Few laboratories have developed imaging agents for positron emission tomography (PET) imaging, and imaging using other modalities such as optical imaging has been virtually unexplored.33 Although PET is a great imaging technique for clinical imaging and translational research due to high sensitivity and no limitation in tissue penetration, the spatial resolution is comparatively low.34 Optical imaging is widely used for biomedical imaging due to its high sensitivity and resolution, as well as low instrument cost.35 In clinical settings, optical imaging is a promising technology for intraoperative guidance36–38 and optical biopsies.39,40 Near-infrared (NIR) fluorescent dyes are typically used as the fluorophores for in vivo imaging applications because of the relatively deep tissue penetration and negligible autofluorescence in the NIR region (650 to 900 nm).41 In our previous study, we reported the first -targeted NIR fluorescent probe, NIRmbc94, which was synthesized by coupling a conjugable pyrazole-based ligand, mbc94, with an NIR fluorescent dye, IRDye800CW.42 We selected the pyrazole structure to develop mbc94 because SR144528, a pyrazole molecule, is a well-known selective inverse agonist with subnanomolar binding affinity and well-characterized biology.43 NIRmbc94 was successfully used to image in a transfected mouse malignant astrocytoma delayed brain tumor (DBT) cell line, -mid DBT, that expresses at endogenous levels.44 Later on, NIRmbc94 was also used to select specific ligands by high-throughput screening in the receptors’ native environment.45 Recently, we reported the first in vivo optical imaging study using another NIR fluorescent probe based on mbc94, NIR760-mbc94, which has an NIR dye (NIR760) with easy synthesis, and high fluorescence quantum yield, stability, and molar extinction coefficient.46 In this study, we developed a novel -targeted NIR fluorescent probe based on a quinolone structure. Quinolone-based molecules were recently reported as highly selective ligands with binding affinities as high as 0.2 nM.47 To develop the quinolone-based probe (NIR760-Q), we synthesized a novel conjugable ligand based on the quinolone structure, followed by bioconjugation with the NIR760 dye. To demonstrate the translational potential of this new probe, NIR760-Q was used to image in Jurkat human acute T-lymphoblastic leukemia cells that naturally overexpress . To our best knowledge, this is the first -targeted cellular imaging of human cancer cells that naturally overexpress the target receptor. 2.Methods and Materials2.1.Synthesis of the -Targeted NIR Probe, NIR760-QThe solvents used are of American Chemical Society (ACS) or high-performance liquid chromatography (HPLC) grade. The dye NIR760 and compound 3 were synthesized according to the method reported previously.46,48,49 A Biotage (Charlotte, North Carolina) microwave reactor (model: US/JPN 356007) was employed for the synthesis of compound 3. Flash column chromatography was run through a Teledyne Isco (Lincoln, Nebraska, combiflash RF) purification system with silica gel (standard grade, 60A, Sorbtech, Norcross, Georgia) or C18-reversed phase silica gel (20 to 40 μm, RediSepRf, Lincoln, Nebraska). and NMR spectra were recorded on a Bruker Avance (Billerica, Massachusetts) III 400-MHz NMR instrument. Mass spectra were recorded on a Waters (Milford, Massachusetts) LCT Premier mass spectrometer. UV/Vis spectra were recorded on a Cary (Santa Clara, California) 100 Bio UV-Vis spectrophotometer, and fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer. Compound 4. Under argon protection, sodium ascorbate (79 mg, 0.4 mmol) in water (1 mL) and 11-azido-3,6,9-trioxaundecan-1-amine (48 μL, 0.24 mmol) followed by copper (II) sulfate (29 mg, 0.2 mmol) in water (0.5 mL) were added to a solution of compound 3 (83 mg, 0.2 mmol) in ethanol (10 mL). The resulting reaction mixture was stirred at room temperature for 2 h and then poured into water (10 mL), followed by extraction with ethyl acetate (). The organic layer was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using (100/10/1) as the eluent. Compound 4 (0.103 g, 81%) was obtained as a colorless oil. NMR (): (s, 1 H), 8.67 (s, 1H), 8.61 (s, 1 H), 8.38 (d, 1 H, ), 8.26 (s, 1 H), 7.56 (d, 1 H, ), 4.64 (br.s, 2 H), 4.22 (t, 2 H, ), 3.93 (br.s, 2 H), 3.56-3.69 (m, 12 H), 2.13 (br.s, 6 H), 2.08 (br.s, 3 H), 1.84–1.87 (m, 2 H), 1.65-1.73 (m, 6 H), 1.33–1.35 (m, 4 H), 0.87 (t, 3 H, ). NMR (): , 163.53, 147.31, 146.12, 138.48, 130.28, 128.06, 127.85, 123.40, 122.14, 116.89, 112.87, 70.45, 70.53, 69.57, 54.47, 51.71, 50.60, 50.51, 41.88, 36.56, 29.55, 28.84, 28.74, 22.26, 13.88. MS (ESI): calculated for [] 635.39, found 635.02. NIR760-Q. A mixture of NIR760 (17 mg, 19 μmol), HBTU (11 mg, 29 μmol), and HOBt (3.8 mg, 28 μmol) in dry dimethylformamide (DMF) (1 mL) was stirred under argon at room temperature for 5 min. N,N-diisopropylethylamine (DIEA) (5 μL, 29 μmol) was then added and the resulting mixture was stirred for another 10 min. Next, compound 4 (12 mg, 19 μmol) in anhydrous DMF (2 mL) was added to the dye solution. The resulting mixture was stirred at room temperature in the absence of light for 20 h. The solvent was then removed by rotary evaporation and the resulting solid was purified by C18-reversed phase column chromatography using (20% MeOH to 75% MeOH) as the eluent resulting in a relatively pure probe. The green solid was further purified by preparative HPLC using a Phenomenex Jupiter C-4 column () at a flow rate of . Flow A was 0.1% triethylamine (TEA) in water and flow B was 0.1% TEA in acetonitrile. The elution method started with a linear gradient from 30% to 70% B over 20 min, then from 70% to 100% B over 10 min, held at 100% B for 3 min, and finally returned to 30% B over 10 min. After being dried by lyophilization, NIR760-Q (7.1 mg, 24%) was obtained as a green solid. NMR (-DMSO): (s, 1 H), 8.77-8.78 (m, 2 H), 8.76 (s, 1 H), 8.70 (t, 1 H, ), 8.28 (dd, 1 H, & 8.8 Hz), 8.12 (d, 2 H, ), 8.00 (d, 1 H, ), 7.57–7.59 (m, 4H), 7.36 (d, 2 H, ), 7.33 (d, 2 H, ), 7.03 (d, 2 H, ), 6.41 (d, 2 H, ), 4.60 (t, 2 H, ), 4.46 (t, 2 H, ), 4.26 (br.s, 4 H), 3.90 (t, 2 H, ), 3.55-3.59 (m, 12 H), 2.71 (br.s, 4 H), 2.55 (t, 4 H, ), 2.07 (br.s, 9 H), 1.90-1.97 (m, 6 H), 1.79 (br.s, 2 H), 1.67 (s, 6 H), 1.30-1.32 (m, 4 H), 1.08 (s, 12 H), 0.84 (t, 3 H, ). NMR (-DMSO): , 171.35, 165.55, 162.79, 160.43, 147.83, 146.96, 145.17, 144.92, 142.20, 141.77, 139.97, 138.31, 133.76, 131.51, 129.94, 129.36, 127.62, 127.59, 127.43, 126.19, 122.52, 122.15, 119.65, 111.71, 110.14, 101.03, 69.75, 69.71, 69.62, 68.87, 68.58, 50.61, 49.73, 48.13, 47.82, 42.69, 41.70, 38.28, 36.08, 28.91, 28.44, 28.03, 27.03, 24.34, 23.26, 21.78, 13.87. MS (ESI): calculated for [M] 1560.57, found 1560.35. 2.2.Reverse Transcription Polymerase Chain Reaction (RT-PCR)Jurkat cells were seeded into a T75 flask and cultured at 37°C for 72 h. Total ribonucleic acid (RNA) was extracted from Jurkat cells using RNAzol Reagent (Invitrogen, Grand Island, New York). Single strand cDNA was synthesized from total RNA using the SuperScript III first-strand synthesis system (Invitrogen) for reverse transcription polymerase chain reaction (RT-PCR). Primers of were obtained from Integrated DNA Technologies (Coralville, Iowa). The sequences used for were 5’ccatggaggaatgctgggtg3’ and 5’tcagcaatcagagaggtctag3’. Amplifications were run using Platinum® Taq DNA polymerase (Invitrogen) and consisted of 36 cycles of 30 s at 95°C, 60 s at 59°C, and 60 s at 69°C. 2.3.In Vitro Saturation Binding Assay of NIR760-QWe carried out intact cell saturation binding assay to determine binding affinity of NIR760-Q to . Briefly, Jurkat cells were seeded into 96 well optical bottom plates (). Cells were incubated for 30 min with an increasing concentration (0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 80.0, and 160.0 nM) of NIR760-Q at 37°C. For nonspecific binding measurements, 1 μM (final concentration) of the blocking agent 4-quinolone-3-carboxamide (4Q3C, Cayman Chemical, Ann Arbor, Michigan) was added with NIR760-Q to each well, while for total binding measurements, no blocking agent was added. Cells were then rinsed with serum free medium and the fluorescence intensity at 790 nm (relative fluorescence units) was recorded with a Synergy H4 Hybrid Multi-Mode Microplate Reader. DRAQ-5, a commonly used cell nuclear DNA labeling dye, was used to normalize cell numbers based on a fluorescence intensity at 690 nm (exited at 650 nm) using the protocol provided by the manufacturer. The specific binding was obtained by the subtraction of nonspecific binding from total binding. The dissociation constant () and receptor density () were estimated from the nonlinear fitting of specific binding versus NIR760-Q concentration using Prism software (GraphPad Prism 6.01, San Diego, California). 2.4.Cell Fluorescent Imaging of NIR760-QJurkat cells were treated with 5 μM of NIR760-Q or NIR760 at 37°C for 30 min, with or without coincubation with 10 μM of 4Q3C as the blocking agent. After being washed three times with serum free medium, cells were fixed with 4% paraformaldehyde/PBS for 20 min at room temperature. The cell nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature. Cells were mounted and then imaged using a Zeiss (Thornwood, New York) Axio Observer fluorescent microscope equipped with the ApoTome 2 imaging system. NIR760-Q or NIR760 fluorescence images were captured using an NIR camera with ICG filter set (excitation/emission: 750 to to 875 nm). Nuclear images were obtained with a DAPI filter set (excitation/emission: 335 to to 470 nm). Differential interference contrast (DIC) images were obtained through Trans light DIC. A multiplate reader system (Synergy H4 Hybrid) was used for the quantitative binding assay measurements. Cells were divided into four groups: (1) Jurkat cells treated with 5 μM of NIR760-Q at 37°C for 30 min, without blocking agent; (2) Jurkat cells treated with 5 μM of NIR760-Q, together with 10 μM of 4Q3C as the blocking agent at 37°C for 30 min; (3) Jurkat cells treated with 5 μM of free NIR760 dye without targeting moiety at 37°C for 30 min; and (4) wild type DBT-cells (-) treated with 5 μM of NIR760-Q at 37°C for 30 min. To prepare for the measurements, DBT-cells were seeded into 96-well optical plates 24 h before the treatment. Jurkat cells were grown to 90% confluence in T75 flasks, harvested, and seeded into 96-well optical plates (). Since most Jurkat cells were in suspension, the 96-well plates were centrifuged before 5 μM of NIR760-Q or NIR760 with or without 4Q3C were added in culture medium. After being incubated for 30 min, cells were washed three times with serum free medium. A Synergy H4 Hybrid Multimode Microplate Reader was used to record fluorescence intensity 790 nm (excitation at 740 nm). Assays for each group were triplicated. 3.Results and DiscussionThe design of NIR760-Q has the following considerations: (1) The quinolone structure was chosen as the targeting moiety because certain quinolone molecules, such as 4-quinolone-3-carboxamide, were recently reported to have high selectivity and binding affinity ( as high as 0.2 nM);47 (2) A terminal amino group was introduced to the targeting moiety to allow for universal conjugation with various signaling moieties, such as fluorescent dyes (for optical imaging) and metal chelators (for PET, single photon emission-computed tomography, and magnetic resonance imaging); (3) NIR760 was selected as the fluorophore for NIR fluorescence imaging, which has high fluorescence quantum yield, stability, and molar extinction coefficient and can be synthesized with only two steps.46 In addition, the four sulfonate groups on NIR760 introduce high hydrophilicity to the imaging probe; (4) A di(ethylene glycol) linker between the targeting moiety and NIR760 dye provides ether oxygens as hydrogen bond acceptors that can potentially facilitate the binding.50 To synthesize NIR760-Q (Fig. 1), we first prepared quinolone compound 3 through 7-steps reactions using previously reported methods.48,49 The following click reaction between compound 3 and 11-azido-3,6,9-trioxaundecan-1-amine provided the ligand 4 with a terminal amine group, which was conjugated with fluorescent dye NIR760 to yield the desired probe, NIR760-Q. Upon the synthesis of NIR760-Q, absorption and emission spectra were collected and compared with NIR760 as shown in Fig. 2. NIR760 has intense NIR absorption and emission in water with peaks at 760 and 781 nm, respectively. After conjugation with ligand 4, the dye showed redshifted absorption and emission spectra. The maximum absorption of NIR760-Q is located at 768 nm with a high molar extinction coefficient of , which is comparable with that of NIR760 ().46 NIR760-Q also exhibits the intense fluorescence centered at 787 nm with a high quantum yield (16.5% in water), which is roughly 60 times higher than that of indocyanine green (ICG) ( in water51). ICG is the only Food and Drug Administration-approved NIR fluorescent dye and typically serves as the gold standard for NIR fluorescence imaging. Fig. 2The absorption and emission spectra of NIR760 (solid) and NIR760-Q (dash) in at a concentration of 1.0 μM. (). ![]() We recently reported a -targeted NIR fluorescent probe, NIR760-mbc94, which was successfully used to image cells and tumors.46 NIR760-Q and NIR760-mbc94 share the same fluorescent dye, NIR760, while the targeting molecules are based on quinolone and pyrazole structures, respectively. Compared to NIR760-mbc94, NIR760-Q has similar maximum absorption (768 versus 766 nm) and emission (787 versus 785 nm) wavelengths, but higher molar extinction coefficient ( versus in water) and quantum yield ( versus 15.2% in water). The enhanced absorption and emission near 800 nm, and excellent water solubility render NIR760-Q as an outstanding fluorescent probe for biomedical imaging. We selected Jurkat human acute T-lymphoblastic leukemia cells to test the binding affinity and in vitro imaging potential of NIR760-Q. In our previous -targeted imaging studies,42,52 we used a transfected mouse malignant astrocytoma DBT cell line, -mid DBT, that expresses at endogenous levels.44 Along with -mid DBT cells, we used WT DBT cells as the -control cells. Although CB2-DBT and WT-DBT cells are an excellent pair for studying targeting specificity, these cells are mouse cell lines and is not naturally expressed. To demonstrate the translational potential of the developed -targeted probe, we used Jurkat cells that have been reported to naturally express .26 Before Jurkat cells were used for imaging, we verified their expression using RT-PCR. RT-PCR was performed using primers for the human receptor. As shown in Fig. 3, a positive band at 1415 base pairs was detected, indicating positive mRNA expression of in Jurkat cells. Fig. 3mRNA expression in Jurkat cells was assessed by RT-PCR. The band at 1415 base pairs (bp) corresponds to . ![]() To determine NIR760-Q’s binding affinity to in living Jurkat cells, we used an in vitro saturation binding assay to measure the equilibrium dissociation constant and the maximum specific binding. A large excess of 4Q3C was added to a parallel set of cells to saturate receptor binding sites and account for nonspecific binding. Figure 4 shows a representative saturation binding curve. NIR760-Q binds to with a of nM and of . Fig. 4In vitro saturation binding assay of NIR760-Q using intact cells. (a) Total binding data (solid) were represented as relative fluorescent units (RFU) on the -axis as a function of NIR760-Q concentration (-axis) in the absence of 4Q3C (blocking agent). Nonspecific binding data (dash) were represented as RFU as a function of NIR760-Q concentration in the presence of 4Q3C. (b) Specific binding data were represented as RFU (-axis) as a function of NIR760-Q concentration (-axis). Data in -axis were obtained by subtracting the nonspecific binding data from the total binding data. The dissociation constant () and receptor density () were estimated from the nonlinear fitting of the specific binding versus the concentration of NIR760-Q using Prism software. Each data point represents the based on triplicate samples. ![]() The targeting specificity and imaging potential of NIR760-Q was evaluated using Jurkat cells that were incubated with 5 μM of NIR760-Q or NIR760. For blocking studies, Jurkat cells were treated with 5 μM of NIR760-Q and 10 μM of 4Q3C. We observed strong fluorescence signal from cells incubated with NIR760-Q, which primarily localized in the cytoplasm [Fig. 5(a)]. In contrast, no significant fluorescence signal was seen from cells incubated with the same concentration of free dye (NIR760) control. Moreover, challenged cells treated with both NIR760-Q and 4Q3C showed lower fluorescence signal than unchallenged cells. These results indicate specific binding of NIR760-Q to the target receptor. Fig. 5NIR760-Q specifically binds to in Jurkat cells. Jurkat cells were incubated for 30 min with 5 μM of NIR760-Q or free NIR760, with or without 10 μM of blocking agent 4Q3C. Cells were then washed three times with serum free medium before imaging. (a) Fluorescence imaging using a Zeiss Axio Observer fluorescent microscopy with ApoTome 2 imaging system. From left to right: ICG filter (red), ICG filter (red) + DAPI filter (blue) merged and differential interference contrast (DIC). Scale bar: 20 μm. (b) Quantitative fluorescent signal was measured with a Synergy H4 Hybrid Multimode Microplate Reader. Wild type DBT-cells were used as a negative control group for comparison. Each data point represents the based on triplicate samples. (***, ****). ![]() To evaluate the targeting specificity of NIR760-Q in a quantitative manner, we used a multiwell plate reader system to perform Jurkat cell binding assays. Similar to the fluorescence imaging study described above, Jurkat cells were divided into three groups: (1) cells incubated with 5 μM of NIR760-Q; (2) cells incubated with 5 μM of free dye; (3) cells incubated with 5 μM of NIR760-Q and 10 μM of 4Q3C. We also used DBT-cells as the -cell line for comparison. As shown in Fig. 5(b), Jurkat cells treated with NIR760-Q showed a 2.8-fold higher fluorescence signal than those treated with NIR760 ( versus , ). Treatment with 4Q3C reduced the uptake of NIR760-Q in Jurkat cells by 40% (from to , ). In addition, -WT DBT cells treated with NIR760-Q showed 40% less fluorescence signal than Jurkat cells ( versus , ). These results indicate specific binding of NIR760-Q to the target receptor and are consistent with our previous cellular imaging studies using NIR760-mbc94, whose uptake was also blocked by 40% when challenged with a ligand.46 The partial inhibition indicates nonspecific binding of NIR760-Q, which may be due to its net negative surface charge. In a recent study, Choi et al.53 reported that replacing the negatively charged NIR fluorescent dyes with a zwitterionic dye that has no net surface charge significantly reduced the nonspecific binding of NIR fluorescent imaging probes. With a net surface charge of , NIR760-Q is likely to have nonspecific binding. We noticed that the cell uptake of NIR760-Q in DBT-WT cells is higher than that of NIR760 in Jurkat cells. Although both uptakes are due to nonspecific binding, the higher lipophilicity of NIR760-Q than NIR760 could have caused the enhanced level of nonspecific binding. Future study will involve developing zwitterionic probes with reduced nonspecific binding to allow for enhanced imaging contrast at the target site. In addition, we plan to develop a xenograft mouse model using Jurkat cells and evaluate the potential of NIR760-Q and new zwitterionic probes in vivo. 4.SummaryIn summary, we have developed a novel -targeted NIR probe NIR760-Q, which demonstrated specific binding in Jurkat human cells. Compared to our previously reported probe, NIR760-mbc94, NIR760-Q has enhanced absorption and emission and comparable binding affinity and specificity. We also report the first -targeted optical imaging of human tumor cells that naturally express . The combined data indicate that NIR760-Q is a promising imaging probe for -targeted imaging with the potential of translational studies. Such an imaging tool may have great value in elucidating the regulatory role of in various diseases. AcknowledgmentsWe thank Dr. Nephi Stella at the University of Washington for providing DBT cells and technical advice. We also thank Dr. Xiangqun Xie at the University of Pittsburgh for providing support to this project. This work was supported by the startup fund provided by the Department of Radiology, University of Pittsburgh. ReferencesT. Sugiuraet al.,
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BiographyZhiyuan Wu received his MD degree from Shanghai Jiao Tong University School of Medicine, China, and is currently working as a postdoctoral fellow in Dr. Mingfeng Bai’s lab at the University of Pittsburgh. His research interests include molecular imaging of cancer and imaging biomarkers research. He has published more than 40 peer-reviewed journal articles in these fields. Pin Shao is currently a research associate in Dr. Mingfeng Bai’s lab at the Department of Radiology, University of Pittsburgh. She obtained her PhD degree in chemistry from Wuhan University, China, and her research interests are in the development of novel near-infrared fluorescent compounds for optical and/or photoacoustic imaging, as well as drug delivery. Shaojuan Zhang earned her PhD degree at Gunma University, Japan, and is currently a postdoctoral fellow at the University of Pittsburgh, under the mentorship of Dr. Mingfeng Bai. Her research interests are targeted optical imaging and cancer therapeutics using molecular probes. Xiaoxi Ling earned his PhD degree in chemistry from Ohio University and is currently a postdoctoral fellow in Dr. Mingfeng Bai’s Lab at the University of Pittsburgh. His research involves the development of near-infrared fluorescence imaging probes and theranostic agents. Mingfeng Bai is an assistant professor of radiology at the University of Pittsburgh. He received his MSc and PhD degrees in chemistry from Vanderbilt University and BS degree in chemistry from Nankai University, China. His research group is focused on developing novel molecular imaging probes for diagnostic imaging and phototherapy. He has published peer-reviewed journal articles, holds various patents, and has given many presentations nationally and internationally. |