Article pubs.acs.org/molecularpharmaceutics
Tumor Targeting and Pharmacokinetics of a Near-Infrared Fluorescent-Labeled δ‑Opioid Receptor Antagonist Agent, Dmt-TicCy5 Amanda Shanks Huynh,† Veronica Estrella,† Valerie E. Stark,† Allison S. Cohen,† Tingan Chen,‡ Todd J. Casagni,§ Jatinder S. Josan,∥,⊥ Mark C. Lloyd,‡ ,⊥ Joseph Johnson,‡ Jongphil Kim,# Victor J. Hruby,∥ Josef Vagner,¶ and David L. Morse*,† †
Department of Cancer Imaging & Metabolism, ‡ Analytic Microscopy Core, §Department of Comparative Medicine, and Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612, United States ∥ Department of Chemistry, The University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85719, United States ¶ The BIO5 Research Institute, University of Arizona, 1657 East Helen Street, Tucson, Arizona 85721, United States #
S Supporting Information *
ABSTRACT: Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist−fluorescent dye conjugate, as a tumortargeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent. KEYWORDS: fluorescence imaging agent, tumor targeting, delta opioid receptor antagonist, pharmacokinetics, biodistribution, dorsal window chamber, cancer imaging, intravital microscopy, live cell imaging, cancer, uptake
■
above.14−20 The labeling of tumors in vivo by novel cancer targeted peptide agents conjugated with near-infrared fluorescent cyanine dyes, like Cy5, has been shown by our group and others.21−28 Despite the current wide array of ligands that bind remarkably well to opioid receptors, the development and evaluation of novel fluorescently labeled opioid receptor agonists or antagonists is limited. The δ-opioid receptor (δOR) is less well characterized than the other two opioid receptor types (κ- and μ-), which all belong to the G-proteincoupled receptor (GPCR) superfamily.29 Typically, δOR internalization is agonist mediated and can occur through G-
INTRODUCTION Noninvasive fluorescence imaging is becoming an important tool in biomedical research that can be used in the development of cancer targeted diagnostic imaging and therapeutic agents. The recent availability of clinical real-time fluorescence imaging systems has opened the door for the development of cancer targeted fluorescent agents for intraoperative use in tumor margin detection and identification of lymph node involvement. One approach to fluorescence imaging of cancer involves the development of imaging agents that selectively bind to cell surface receptors with high expression on cancer cells and minimal expression on normal non-neoplastic tissue. The growing list of cancer-specific receptors identified includes epidermal growth factor,1−3 folate receptor,4−6 integrins,7,8 and somatostatin receptors.9−13 Currently there are a variety of fluorescent-labeled cancer imaging agents either in development or commercially available that target the receptors listed © 2015 American Chemical Society
Received: Revised: Accepted: Published: 534
October 7, 2015 December 11, 2015 December 29, 2015 December 29, 2015 DOI: 10.1021/acs.molpharmaceut.5b00760 Mol. Pharmaceutics 2016, 13, 534−544
Article
Molecular Pharmaceutics
sets were designed using Gene Runner software for Windows v 3.05: forward, 5′-GGTGACCAAGATCTGCGTGTTC-3′, and reverse, 5′-TTCTCCTTGGAGCCCGACAG-3′. The iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad, Cat. No. 170-8893) was used for qRT-PCR. During each experiment, reactions were performed using template without RT mix and without template added as controls. β-Actin (ACTB) was used for normalization.58 The following conditions for thermocycling were used: Stage 1 was held at 50 °C for 10 min for cDNA synthesis; stage 2 was held at 95 °C for 5 min for reverse transcriptase inactivation; stage 3 cycled 40 times through two temperatures for PCR amplification, starting with 95 °C for 10 s and Tm for 30 s (Tm is 60 °C for ACTB and 62 °C for OPRD1); and stage 4 included a melt curve for quality control, starting at 40 °C and ending at 95 °C (increasing by 0.2 °C each cycle). Values were calculated as expression = 2−Ct(OPRD1)/ 2−Ct(ACTB) × 1000. Expression of OPRD1 was normalized to the expression of β-actin in each cell line. Each experiment was performed in triplicate. In Vitro Receptor Expression Confirmation and Receptor Number Determination. To verify the expression of δOR on HCT 116/δOR cells and no expression on the HCT 116 parental cell line, TRF saturation binding assays were performed as previously described.21,55,59−61 The number of δopioid receptors expressed on the cell surface of HCT 116/ δOR cells was calculated using an adapted version of the binding assay, as previously described.60 To calculate the number of receptors per cell, standard curves of the relationship between fluorescence intensities and ligand concentrations were generated. The standard curves were then used to determine the amount of ligand present at the Bmax obtained in the saturation binding assay. The average number of cells per well at the end of the assay was calculated. To determine the receptor number, the following equation was used: (Eu amount for Bmax (mole)/av cell number per well) × 6.023 × 1023 = receptor number per cell. In Vitro Live Cell Epifluorescence Microscopy. Live cell epifluorescence microscopy was used to evaluate cellular uptake of the Dmt-Tic-Cy5 agent in vitro. HCT 116/δOR cells were grown on glass-bottom plates (World Precision Instrument FluoroDish, 35 mm). Cellular uptake of the Dmt-Tic-Cy5 agent was monitored by acquiring fluorescence images from 0 to 24 h with a Zeiss Z1 observer microscope (Carl Zeiss Inc., Germany) using a 40× oil objective, Cy5 filter cube, and a MRm3 CCD camera with an exposure time of 1.5 s. The microscope is equipped with a fully enclosed incubation chamber set to 37 °C and 5% CO2. AxioVision v4.8.2 software was used to obtain the images, and the time-lapse sequences and images are acquired using the same parameters over the time course so that no pixels are saturated and direct comparisons can be made among the images. The cells were rinsed once with PBS, and Dmt-Tic-Cy5 (2.5 nM in FluoroBrite DMEM medium) was added. Immediately following ligand addition, the plate was placed on the stage in the incubation chamber and images were acquired at 30 s intervals for 15 min. After 15 min, the medium was removed and the cells were rinsed once with FluoroBrite DMEM medium. The cells were incubated in FluoroBrite DMEM medium for the remainder of the imaging session. The mark and find feature was used to image 4 random fields of view every 10 min for 24 h starting approximately 30 min post ligand addition. The experiment was performed in triplicate on consecutive days.
protein-dependent and G-protein-independent pathways, which involve phosphorylation, β-arrestin, or clathrin-coated vesicles.30 No internalization of δORs has been observed with antagonists. In 2012, Granier et al. reported the crystal structure of murine δOR bound to naltrindole,31 one of the few highly potent and δ-selective antagonists (e.g., naltriben,32,33 naltrindole,32,34,35 and Dmt-Tic).36−39 δOR-targeted nuclear medicine agents have been developed for positron emission tomography (PET) and single-photon emission computed tomography (SPECT).40,41 To the best of our knowledge, our Dmt-Tic-Cy5 and Dmt-Tic-IR800 are the only accounts of fluorescent δOR antagonist targeted peptide agents that exhibit low nanomolar high binding affinity.21,42 The δOR has been reported to be expressed in various human cancers, such as lung, liver, and breast.42−46 Even though the full mechanism is not yet known, δOR has a role in driving tumor progression.44,47−49 Recent studies indicate that δOR inhibition can decrease cellular proliferation and induce apoptosis of tumor cells.47,50,51 Hence, δOR antagonists may have therapeutic utility. A fluorescently labeled δOR targeted antagonist may have prolonged tumor associated fluorescence compared to agonist-based agents due to slower receptor off rates52 and slower cellular uptake kinetics, leading to delayed timing of lysosomal degredation.53 Herein, we investigate the potential of using Dmt-Tic-Cy5 as a tumor targeted fluorescent-labeled molecular agent by characterizing cellular uptake, tumor targeting, and pharmacokinetics (PK) of this agent in vivo using mouse tumor models with and without expression of the δOR and acquisition of fluorescence images over time following intravenous (iv) administration.
■
EXPERIMENTAL SECTION Fluorescent δOR-Targeted Agent Synthesis. Dmt-TicCy5 (Dmt-Tic-Lys(Cy5)-OH was synthesized by solid-phase synthesis as previously described.21 Cell Culture. The parental HCT 116 (ATCC #CCL-247) cell line was purchased from ATCC (Manassas, VA). The HCT 116 colon cancer cells were genetically engineered to highly overexpress the δ-opioid receptor using pcDNA-δOR15 vector containing a truncated δOR lacking the final 15 C-terminal amino acids, and are hereon termed HCT 116/δOR.54,55 HCT 116 cells were engineered to express red fluorescent protein (RFP), hereon termed HCT-116/RFP, and were used as the negative, non-δOR-expressing tumor control for the intravital imaging experiments.56 Upon completion of experiments, cell lines were authenticated using short tandem repeat (STR) DNA typing according to ATCC’s guidelines.57 The cell lines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% normal calf serum (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin solution (Sigma, St. Louis, MO). mRNA Expression Using Quantitative Real-Time RTPCR (qRT-PCR). The relative mRNA expression levels of OPRD1, the gene that encodes the δOR, were determined using quantitative real-time RT-PCR (qRT-PCR). RNA extractions were performed on cell lines using the RNeasy Mini Kit (Qiagen, Cat. No. 74104) following the manufacturer’s instructions, which include the DNase digestion steps. RNA concentration and purity (A260/A280 ratio of ∼2.0) were determined by using the NanoDrop spectrophotometer, ND1000 (Wilmington, DE). qRT-PCR was performed using the Smart Cycler (Cephid, Sunnyvale, CA). OPRD1 specific primer 535
DOI: 10.1021/acs.molpharmaceut.5b00760 Mol. Pharmaceutics 2016, 13, 534−544
Article
Molecular Pharmaceutics
removing the animals and repeating the measurements and was subtracted from each image. Autofluorescence was determined by drawing identical ROIs on the instrument background subtracted Cy5 fluorescence image and corresponding image acquired prior to agent administration, and subtracting the autofluorescence values from the postadministration image. Pharmacokinetics. We chose to evaluate the pharmacokinetics (PK) of the Dmt-Tic-Cy5 agent in vivo over a time course (0−168 h) after the administration of a single dose of 4.5 nmol/kg in a volume of 100 μL. δOR− and δOR+ engineered HCT 116 cells (8 × 106) were bilaterally xenografted into left and right flanks of athymic nude female mice, respectively. Tumor volume was determined with calipers using the formula volume = (length × width2)/2. Mice were ready for fluorescence imaging after a minimum size of 500 mm3. Tumor size ranged from 500 to 800 mm3. Mice were imaged in vivo over the time course (3 min, 9 min, 15 min, 30 min, 45 min, 1.5 h, 3 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h, 96 h, 168 h) using the IVIS 200 system and Cy5 filter sets. The data were analyzed after performance of instrument background subtractions and tissue autofluorescence subtraction performed using a preinjection acquisition and identical ROIs as described above. The fluorescence signals were quantified using Living Image Software (PerkinElmer Xenogen Caliper Life Sciences, Hopkinton, MA). ROIs were drawn, and mean surface radiance normalized for heterogeneity of stage lighting (efficiency units) was used. Uptake and clearance parameter estimations were performed using analysis tools available in GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com. Biodistribution Studies. To confirm the tumor selectivity and tissue distribution of the Dmt-Tic-Cy5 agent in the PK studies, both in vivo and ex vivo fluorescence imaging acquisitions were performed on an additional group of mice to determine the biodistribution (BD) of Dmt-Tic-Cy5. Mice were ready for fluorescence imaging after a minimum size of 500 mm3. Tumor sized ranged from 500 to 800 mm3. At select time points (0, 7.5 min, 15 min, 45 min, 3 h, 6 h, 24 h, 48 h) postadministration of 4.5 nmol/kg Dmt-Tic-Cy5 agent, mice were in vivo fluorescence imaged and euthanized, and tumors and organs (spleen, pancreas, liver, kidneys, GI tract, heart, lungs, and tumors) were harvested and imaged ex vivo to determine the agent’s BD. Imaging was performed using the IVIS 200 system and Cy5 filter sets. The data were analyzed in the same manner as in the PK study described above. Intravital Tumor Cell Uptake Studies. Intravital confocal microscopy was used to evaluate agent circulation, extravasation, and cellular uptake of the Dmt-Tic-Cy5 agent in vivo using our previously described mouse dorsal skinfold window chamber (DWC) model (Supporting Figure S1).56,62,63 Cellular internalization of the Dmt-Tic-Cy5 agent was monitored from 0 to 24 h using confocal fluorescence microscopy with a 25× lens and an acquisition rate of 3570 pixels/min. Initially, images were continuously acquired for 30 min postinjection of 45 nmol/kg Dmt-Tic-Cy5 agent via tail vein and at several other time points up to 24 h. To determine extravasation rates, the mean fluorescence intensity was measured in the vessel interior and in the tumor tissue adjacent to the vessel by drawing ROIs on the confocal image sets from various time points using ImagePro v6.2. For visualization purposes, the units of intensity were pseudocolored using a rainbow scale in which the dynamic range value was 0−255 for an 8 bit image, where 0 is blue and 255 is red. To determine
AxioVision v4.8.2 software was used to analyze the images. To determine cell uptake rates, the fluorescence was measured in the cytoplasm (inside) and on the cell membrane (outside) by drawing regions of interest (ROIs) on the images from various time points. The cells were tracked throughout the image sequence. For quantification, a ROI was drawn outside the cell membrane to measure the total fluorescence for the whole cell. Another ROI was drawn immediately inside the cell membrane to measure the fluorescence for the cytoplasm (inside). For each region, the fluorescence was determined by multiplying the average fluorescence intensity by the area. The fluorescence on the cell membrane (outside) was determined by subtracting the fluorescence of the cytoplasm (inside) from the total fluorescence. The difference of fluorescence for the cytoplasm (inside) to fluorescence on the cell membrane (outside) was calculated for each cell at each time point. The ratio inside/outside for individual cells was averaged for each day at each time point. Photobleaching was observed due to multiple acquisitions in the same field of view. However, the use of inside/outside ratios for each individual image minimized the effects of photobleaching, allowing for comparison among the different acquisitions over time. The number of cells analyzed per day was between 11 and 25. GraphPad Prism 6 was used to plot the results. Each data point indicates the average of 3 days, with error bars indicating the standard deviation. Animals. All procedures were in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the Institutional Animal Care and Use Committee, University of South Florida, under the approved protocols R3715 and R4002. Immunocompromised mice were housed in a clean facility with special conditions that include HEPA filtered ventilated cage systems, autoclaved bedding, autoclaved housing, autoclaved water, irradiated food, and special cage changing procedures. Mice were handled under aseptic conditions including the wearing of gloves, gowns, and shoe coverings. Mice were anesthetized by inhaled isofluorane gas and remained anesthetized for the minimum amount of time required for imaging studies, ranging from 3 to 45 min at a time. Dose Determination for in Vivo Imaging Studies. Cells (8 × 106) were xenografted into the left (HCT 116) and right (HCT 116/δOR) flanks of female nu/nu mice (Harlan, Indianapolis, IN). After 1 week, tumors were ready for imaging. A range of dosages from 0.005 to 140 nmol/kg of Dmt-Tic-Cy5 were administered via tail iv injection volume of 100 μL. Animals were imaged immediately to check for successful injection. Follow-up imaging was performed 24 h postinjection. Imaging was performed using an IVIS Imaging System, 200 Series (PerkinElmer Xenogen Caliper Life Sciences, Hopkinton, MA). Excitation (615−665 nm) and emission (695−770 nm) filters were used in wavelength ranges suitable for in vivo excitation and detection of emitted light of Cy5 dye. Acquisition times ranged from 4 to 10 s in order to keep intensity counts above a minimum of 15,000 but below saturation values of ∼60,000. Results are displayed in efficiency units in which the fluorescence surface radiance values (photons/s/cm2/sr) within an image are normalized using a stored reference image that represents the variation in excitation light intensities across the stage, so that images acquired at different times and locations on the stage can be directly compared. Instrument background was determined by 536
DOI: 10.1021/acs.molpharmaceut.5b00760 Mol. Pharmaceutics 2016, 13, 534−544
Article
Molecular Pharmaceutics cell uptake rates of agent, the average pixel intensities were determined using Definiens Developer v2.0. The units of intensity are the dynamic range value 0−255 for an 8 bit image, where 0 is black and 255 is pure white. Since the δOR− images had surrounding vasculature for extravasation studies, ROIs were drawn that did not include vessels for this analysis. δOR+ images were avascular, so pixel intensity values from the entire image were averaged. For presentation purposes, the contrast was uniformly adjusted among the images and the grayscale agent related fluorescence signal is depicted in rainbow scale by pseudocolor. However, only the original unmodified images were used for the analysis. Statistics. Data are represented as mean ± SD. To determine statistical significance, Satterthwaite t-test (2 sample unequal variance parametric test) was employed for three reasons: (1) it is more powerful than nonparametric tests for small sample size studies, (2) the strength of the association is of interest, and (3) two variances are not necessarily the same. The Anderson−Darling test was used to evaluate the normality.
■
RESULTS Receptor Number Determination. For this study, HCT 116 colon carcinoma cells engineered to express the δOR (HCT 116/ δOR) were used as a target receptor positive in vitro cellular and in vivo tumor xenograft model and the parental HCT 116 cells were used as the target negative control.21 To characterize δOR expression levels in these cell lines, quantitative real-time RT-PCR (qRT-PCR) was used to determine the relative mRNA expression levels of OPRD1, the gene that encodes the δOR. The expression of OPRD1 was negligible in the HCT 116 parental cells and was significantly higher in the HCT 116/δOR cells (p-value
