1.

Introduction

The type 2 cannabinoid receptors (${\mathrm{CB}}_2\mathrm{R}$) are G protein-coupled receptors (GPCR) that belong to the endocannabinoid signaling system. They play a fundamental role in the regulation of a variety of physiopathological processes involving the immune system, including diseases that affect the cardiovascular and respiratory systems, bone remodeling and gastrointestinal, and liver diseases.¹ Under healthy conditions, high-${\mathrm{CB}}_2\mathrm{R}$ expression is present only in B cells of the immune system that are localized in spleen and lymph nodes.² In a number of additional cell types under disease conditions, ${\mathrm{CB}}_2\mathrm{R}$ expression is greatly up-regulated.³ The richness of ${\mathrm{CB}}_2\mathrm{R}$ involvement has positioned the receptor as an attractive therapeutic target for treating an array of pathologies, such as cancers,^4,5 neurodegenerative diseases,^2,6 inflammation,^7,8 pain,⁹ osteoporosis,¹⁰ immunological disorders,^11–13 and drug abuse.¹⁴ As such, there has been an explosion of research over the past 12 years that focused on the biology and therapeutic promises of ${\mathrm{CB}}_2\mathrm{R}$. However, the precise role of ${\mathrm{CB}}_2\mathrm{R}$ in the regulation of diseases remains unclear.¹⁵ The ability to specifically image ${\mathrm{CB}}_2\mathrm{R}$ would contribute to develop reliable ${\mathrm{CB}}_2\mathrm{R}$-based therapeutic approaches with a better understanding of the mechanism of ${\mathrm{CB}}_2\mathrm{R}$ action in these diseases.

Molecular imaging (MI) has emerged as a valuable tool for the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems.¹⁶ To overcome the current challenge of identifying ${\mathrm{CB}}_2\mathrm{R}$, we therefore set out to develop MI tools that can specifically label ${\mathrm{CB}}_2\mathrm{R}$, quantify the expression level and image the receptor in a living system. One clear strength of ${\mathrm{CB}}_2\mathrm{R}$-targeted MI agents is the potential for high-contrast imaging. ${\mathrm{CB}}_2\mathrm{R}$ expression is high only in spleen and lymph nodes, and low—or even undetectable—in brain, thyroid, retina, placenta, skeletal muscle, kidney, liver, adrenal gland, heart, prostate, and ovary.^17,18 This allows for imaging of physiological ${\mathrm{CB}}_2\mathrm{R}$ activity in the immune system with low background elsewhere. In certain pathological conditions, ${\mathrm{CB}}_2\mathrm{R}$ expression is greatly up-regulated in the diseased cells. For example, ${\mathrm{CB}}_2\mathrm{R}$ is over-expressed in various types of cancers,¹⁹ endometrial inflammation,²⁰ and atherosclerotic plaques.²¹ ${\mathrm{CB}}_2\mathrm{R}$ expression also increases in diseased central nervous system cells, including astrocytomas,²² activated microglia and neurons,^14,15,23 astrocytes of Alzheimer’s disease,²⁴ as well as T-lymphocytes, microglia, and astrocytes in multiple sclerosis.²⁵ Because ${\mathrm{CB}}_2\mathrm{R}$ expression is nearly absent in these areas under basal conditions, and increases as much as 100-fold in diseases,³ ${\mathrm{CB}}_2\mathrm{R}$ imaging can achieve a high-imaging contrast in diseased tissues. Accordingly, ${\mathrm{CB}}_2\mathrm{R}$ is a promising target for MI applications.

${\mathrm{CB}}_2\mathrm{R}$ imaging remains a largely unexplored field. Few ${\mathrm{CB}}_2\mathrm{R}$ contrast agents that can identify the receptor specifically at the cellular scale have been developed. We recently reported a ${\mathrm{CB}}_2\mathrm{R}$-targeted near-infrared (NIR) fluorescent probe, NIR760-mbc94, which was successfully used to image the receptor in vitro and in vivo.²⁶ Although the specific binding of NIR760-mbc94 to ${\mathrm{CB}}_2\mathrm{R}$ has been demonstrated, the probe also showed nonspecific binding. Specifically, SR144528, a ${\mathrm{CB}}_2\mathrm{R}$ ligand, partially inhibited the uptake of NIR760-mbc94 in vitro (40%) and ex vivo (35%).²⁶ Recently, Dr. Frangioni’s group found that replacing charged NIR fluorescent dyes with a zwitterionic ($\text{net charge}=0$) NIR dye significantly reduced the nonspecific binding of integrin ${\alpha }_v{\beta }_3$-targeted imaging agents.²⁷ Inspired by this report, in this study, we introduced zwitterionic properties into our ${\mathrm{CB}}_2\mathrm{R}$-targeted agents. We found that our zwitterionic ${\mathrm{CB}}_2\mathrm{R}$ probe, ZW760-mbc94, enhanced blocking effect from 40% to 50% in vitro and 35% to 47% ex vivo when compared with NIR760-mbc94. Such improvement may greatly improve the outcome of ${\mathrm{CB}}_2\mathrm{R}$-targeted imaging.

2.

Methods and Materials

3.

Results and Discussion

The structure of the ${\mathrm{CB}}_2\mathrm{R}$-targeted zwitterionic probe, ZW760-mbc94, was designed with the following considerations: (1) We chose mbc94 as the targeting moiety, which was previously reported by us to have specific binding to ${\mathrm{CB}}_2\mathrm{R}$.^3,18,26 (2) We developed a novel zwitterionic NIR dye, ZW760, which has a robust $\mathrm{C}─\mathrm{C}$ linkage at the meso position of the polymethine chain, as compared with the enol ether linkage in the recently reported ZW800 dyes.²⁸ Previous studies indicate that heptamethine cyanine dyes with an enol ether linkage have significant stability issues, which can be overcome by replacing the enol ether with a $\mathrm{C}─\mathrm{C}$ linkage.^26,30 In addition, the carboxylate group on ZW760 allows for universal conjugation to various targeting moieties.

ZW760-mbc94 was synthesized by following the pathway described in Fig. 1. Briefly, we first prepared ZW-Cl and mbc94 using the previously reported methods.^18,28 Next, Suzuki-Miyaura coupling reaction between 4-carboxyphenylboronic acid and ZW-Cl in the presence of $\mathrm{Pd}{({\mathrm{PPh}}_3)}_4$ yielded ZW760. Finally, mbc94 was coupled to ZW760 using an amide-coupling reaction.

Fig. 1

Synthetic pathway of ZW760-mbc94.

Upon the syntheses of ZW760 and ZW760-mbc94, we characterized the spectroscopic properties of these two-fluorescent molecules. When dissolved in methanol or dimethyl sulfoxide (DMSO), ZW760 molecules exist mainly as monomers at a concentration of 1 μM, as indicated by the absorption spectra (Fig. 2). ZW760 showed an intense absorption peak at 786 nm with a weak shoulder at 720 nm in DMSO. The small-shoulder peak is ascribed to the well-known H-type aggregation.³¹ The shape of the ZW760 absorption spectrum in methanol is similar to that in DMSO, except that the absorption peak is blue shifted for 20 nm. ZW760 exhibited strong NIR fluorescence in methanol and DMSO. The fluorescent peak is located at 805 and 786 nm in DMSO and methanol, respectively. When water was used as the solvent, ZW760 showed two major absorption bands at 681 and 756 nm with molar extinction coefficient of $9.6\times {10}^4$ and $8.7\times {10}^4{\mathrm{M}}^{-1}{\mathrm{cm}}^{-1}$, respectively. As discussed above, the strong absorption at 681 nm is ascribed to the H-aggregation of the dye in water. The aggregation of ZW760 in water was further evidenced by the concentration dependent absorption studies. When the concentration of ZW760 increased from $1\times {10}^{-6}$ to $1\times {10}^{-5}\mathrm{M}$ in water, the intensity of the two absorption bands increased simultaneously [Fig. 3(a)]. However, the ratio of absorption at $681\mathrm{nm}/756\mathrm{nm}$ increased greatly [Fig. 3(b)], indicating enhanced H-aggregation at higher concentration. As expected, the fluorescence quantum yields decreased dramatically when concentration increased [Fig. 3(e)]. Due to the reabsorption effect at high concentration,³² the fluorescent peaks are red shifted gradually from 775 to 781 nm [Figs. 3(c) and 3(d)].

Fig. 2

UV–Vis absorption and emission of ZW760 and ZW760-mbc94 in water at a concentration of 1 μM (${\lambda }_{\mathrm{ex}}=700\mathrm{nm}$).

Fig. 3

The concentration-dependent spectra of ZW760 in water. (a) UV–Vis absorption spectra; (b) absorption spectra normalized at 720 nm; (c) emission spectra; (d) the normalized emission spectra (${\lambda }_{\mathrm{ex}}=700\mathrm{nm}$); and (e) the concentration-dependent fluorescence quantum yield change.

Attachment of the targeting moiety to ZW760 significantly reduced the aggregation. As displayed in Fig. 4, ZW760-mbc94 showed an intense absorption band centered at 762 nm in water and the band profile is similar to that of ZW760 in DMSO and methanol. This indicated that ZW760-mbc94 existed mainly as monomers in water at the concentration of 1 μM. The dissociation of the aggregate significantly improved the absorption and emission of ZW760-mbc94. Compared with the free dye ZW760, the absorption peak of ZW760-mbc94 is slightly red shifted to 762 nm and the molar extinction coefficient almost doubled ($9.6\times {10}^4$ and $1.74\times {10}^5{\mathrm{M}}^{-1}{\mathrm{cm}}^{-1}$ for ZW760 and ZW760-mbc94, respectively). The emission peak of ZW760-mbc94 is also red shifted from 775 to 781 nm as compared with ZW760. Importantly, the fluorescence quantum yield of ZW760-mbc94 in water is 15.2%, which is 31% higher than that of ZW760 (11.6%).

Fig. 4

UV–Vis absorption (solid) and emission spectra (dash) of ZW760 (red) and ZW760-mbc94 (black) in water at a concentration of 1 μM (${\lambda }_{\mathrm{ex}}=700\mathrm{nm}$).

Cellular uptake of ZW760-mbc94 was investigated in ${\mathrm{CB}}_2$-mid DBT cells that express ${\mathrm{CB}}_2\mathrm{R}$ at endogenous levels.²⁹ All fluorescence images taken are confocal-like images using a Zeiss Axio Observer fluorescence microscope equipped with the ApoTome 2 imaging system. Figure 5(a) shows fluorescence images of ${\mathrm{CB}}_2$-mid DBT cells and WT DBT-cells (non-${\mathrm{CB}}_2\mathrm{R}$ expressing control) after incubation with 5 μM of ZW760-mbc94, $\mathrm{ZW}760\text{-}\mathrm{mbc}94+4$-Quinolone-3-Carboxamide (4Q3C, blocking agent), or ZW760 (free dye control) for 30 min at 37°C. Strong-fluorescence signal was shown throughout the intracellular cytoplasm of ${\mathrm{CB}}_2$-mid DBT cells treated with ZW760-mbc94 probes. No accumulation in the nucleus was observed. It is noteworthy that the cell membrane did not appear to be the primary location of the probe uptake, which may seem unexpected as ${\mathrm{CB}}_2\mathrm{R}$ belongs to the transmembrane GPCR family. However, recent studies have shown that ${\mathrm{CB}}_2\mathrm{R}$’s distribution was predominantly intracellular in many ${\mathrm{CB}}_2\mathrm{R}$-expressing cell lines.³³ When blocked with 4Q3C, ZW760-mbc94 showed a much lower degree of cellular uptake. Furthermore, no significant fluorescence signal was seen from ${\mathrm{CB}}_2$-mid DBT cells incubated with the free dye (ZW760) control or WT DBT-cells treated with ZW760-mbc94. The combined fluorescence imaging results indicate that ZW760-mbc94 binds to ${\mathrm{CB}}_2\mathrm{R}$ specifically and ${\mathrm{CB}}_2\mathrm{R}$ expression in ${\mathrm{CB}}_2$-mid DBT cells is mainly intracellular.

Fig. 5

ZW760-mbc94 specifically binds to ${\mathrm{CB}}_2\mathrm{R}$ in ${\mathrm{CB}}_2$-mid DBT cells. Four groups: (1) ${\mathrm{CB}}_2$-mid DBT cells treated with 5 μM of ZW760-mbc94 without the blocking agent; (2) ${\mathrm{CB}}_2$-mid DBT cells treated with 5 μM of ZW760-mbc94 together with 10 μM of 4Q3C; (3) ${\mathrm{CB}}_2$-mid DBT cells treated with 5 μM of free ZW760 dye; (4) wild type DBT-cells (${\mathrm{CB}}_2\mathrm{R}$-) treated with 5 μM of ZW760-mbc94. All cells were incubated at 37°C for 30 min and washed three times with serum free medium. (a) Fluorescence images were obtained using a Zeiss Axio Observer fluorescent microscopy with ApoTome 2 imaging system. From top to bottom: ICG filter (red), ICG filter (red) + DAPI filter (blue) merged, differential interference contrast (DIC). Scale: 20 μm. (b) Quantitative fluorescent signal was measured using a Synergy™ H4 hybrid multimode microplate reader. Each data point represents the $\text{mean}\pm \text{standard deviation}$ (SD) based on triplicate samples. (****$p<0.0001$).

As mentioned in Sec. 1, the main purpose of this study is to evaluate whether replacing the charged NIR760 dye, with zwitterionic ZW760 dye would significantly reduce the nonspecific binding of ${\mathrm{CB}}_2\mathrm{R}$-targeted imaging agents. To quantitatively study the specific uptake of ZW760-mbc94, we screened ${\mathrm{CB}}_2$-mid DBT and WT DBT-cells using a multiplate reader. As shown in Fig. 5(b), the uptake of ZW760-mbc94 in ${\mathrm{CB}}_2$-mid DBT was effectively blocked by 4Q3C ($8161.7\pm 211.4$ versus $4106.3\pm 421.0$, $p<0.0001$). This roughly 50% blocking effect is higher than our previous result using NIR760-mbc94 as the imaging probe,²⁶ indicating reduced nonspecific binding. In addition, fluorescence signal from ${\mathrm{CB}}_2$-mid DBT cells treated with free dye is only 6.6% of that with ZW760-mbc94 ($8161.7\pm 211.4$ versus $542.7\pm 154.7$, $p<0.0001$). Furthermore, the uptake of ZW760-mbc94 in WT DBT cells was $\sim 40\%$ lower than in ${\mathrm{CB}}_2$-mid DBT cells ($5061.3\pm 272.6$ versus $8161.7\pm 211.4$, $p<0.0001$). Therefore, the results provided additional evidence for the specificity of ZW760-mbc94, which was improved over the previously reported NIR760-mbc94 probe.

The specific binding of ZW760-mbc94 to ${\mathrm{CB}}_2\mathrm{R}$ was further demonstrated by the in vitro fluorescence saturation binding study. ZW760-mbc94 served as the fluorescent ligand and 4Q3C was used as the blocking agent to account for nonspecific binding. A representative saturation binding curve is shown in Fig. 6. Similar to NIR760-mbc94, ZW760-mbc94 binds to ${\mathrm{CB}}_2\mathrm{R}$ with a nanomolar affinity ($K_d=53.9\pm 13.0\mathrm{nM}$).

Fig. 6

In vitro fluorescence saturation binding assay of ZW760-mbc94. The equilibrium dissociation constant (K_d) and the maximum specific binding (Bmax) were calculated from in vitro saturation-binding assay using Prism software. ${\mathrm{CB}}_2$-mid DBT cells were incubated for 30 min with an increasing concentration of ZW760-mbc94 with or without the blocking agent (4Q3C). Cells were then rinsed with serum free medium and fluorescence at 790 nm was recorded with a Synergy™ H4 hybrid multimode microplate reader. Each data point represents the $\text{mean}\pm \mathrm{SD}$ based on triplicate samples.

To study the targeting specificity of ZW760-mbc94 in living systems, in vivo optical imaging was performed $\sim 10\text{days}$ postinoculation of ${\mathrm{CB}}_2$-mid DBT tumor cells subcutaneously. The tumor sizes in all mice showed no significant difference. Three groups of mice (three in each group) were administrated with ZW760-mbc94, $\mathrm{ZW}760\text{-}\mathrm{mbc}94+4\mathrm{Q}3\mathrm{C}$, and ZW760, respectively. The representative in vivo time-dependent fluorescence images are shown in Fig. 7(a). All images are displayed on the same scale to allow for direct visual comparison among the uptake profiles of ZW760-mbc94, $\mathrm{ZW}760\text{-}\mathrm{mbc}94+4\mathrm{Q}3\mathrm{C}$, and ZW760 dye. The free ZW760 dye showed rather quick clearance profile. Right after the injection, intense fluorescence signal was observed throughout the whole body. Most dye molecules were cleared out only 30 min after injection, and no significant fluorescence signal was observed from the animals after the 1-h PI time point. On the contrary, ZW760-mbc94 showed much slower bioclearance profiles, with or without the blocking agent (4Q3C). Significant fluorescence signal was observed from the whole body even at the last imaging time point (72-h PI).

Fig. 7

In vivo tumor optical imaging and competitive blocking studies. $1\times {10}^6$ ${\mathrm{CB}}_2$-mid DBT cells were subcutaneously implanted into the right flank of 6- to 8-week-old female Nu/nu mice. Experiments with tumor-bearing mice were performed 10 days after injection of tumor cells. To examine tumor ${\mathrm{CB}}_2\mathrm{R}$-targeted imaging, all mice were injected with the following agents dissolved in 100-μL saline via the tail vein: 3 with 10-nmol ZW760-mbc94, 3 with 10-nmol ZW760-mbc94 + 100 nmol 4Q3C, 3 with 10-nmol free ZW760. (a) Mice were anesthetized with 2.5% isoflurane and imaged with a charge-coupled device camera-based bioluminescence imaging system IVIS Lumina XR, at preinjection, 0.02-, 0.5-, 1-, 2-, 4-, 6-, 12-, 24-, 48-, and 72-h postinjection (PI). (b) Time activity curves of the average radiant efficiency of the tumor area among three groups. Radiant efficiency of the tumor area was calculated by the region of interest (ROI) function of Living Image software. (c) Time activity curves of tumor/background ratio among three groups. Radiant efficiency of the tumor area at the right flank of the animal (T) and of the area at the left flank [normal tissue (N)] was calculated by the ROI function of Living Image software. Dividing T by N yielded the contrast between the tumor tissue and the normal tissue. (d) Previously published in vivo imaging results using NIR 760-mbc94 for comparison.²⁶ In vivo images were collected at preinjection, 0.02-, 1-, 2-, 6-, 12-, 24-, 48- and 72-h PI. (*$p<0.05$).

The tumor (located at the right flank) of the mice injected with ZW760-mbc94 showed higher fluorescence signal than that in the blocked animals at almost all time points [Fig. 7(b)], with statistical significance ($p<0.05$) achieved at 4-h PI. The ratio of the fluorescence signal at the tumor area (T) over that at the left leg [normal tissue (N)] was used to calculate the tumor contrast (T/N). For the free dye control mice, intense fluorescent signal was observed throughout the whole body within the first-hour PI without visual tumor contrast, whereas no fluorescence signal was observed anywhere at other time points. For the mice injected with ZW760-mbc94, the T/N ratio gradually increased over time and reached the maximum ($\mathrm{T}/\mathrm{N}=1.96\pm 0.15$) at about 12-h PI [Fig. 7(c)]. Mice treated with $\mathrm{ZW}760\text{-}\mathrm{mbc}94+4\mathrm{Q}3\mathrm{C}$ showed lower T/N ratio at all time points [Fig. 7(c)], with the peak T/N ratio ($\mathrm{T}/\mathrm{N}=1.63\pm 0.22$) at 6-h PI. As shown in Fig. 7(c), significant difference of the T/N ratio between the probe and blocking group was achieved at 48-h (20% blockage effect, $p=0.023$) PI. Because of the signal interference caused by systemic probe uptake at these time points [Fig. 7(a)], these percentages may not represent the true blocking effect. As such, we will use the ex vivo imaging results to evaluate the specificity of ZW760-mbc94 in the living system.

After the last imaging time point (72-h PI), the mice were sacrificed, and the major organs (blood, heart, lung, liver, spleen, pancreas, kidney, muscle from left leg, and brain) were harvested for ex vivo biodistribution analysis [Fig. 8(a)]. The fluorescent intensities in tumor and organs were measured, and the signal contrast (fluorescence signal from tumor or organ/signal from muscle) values are presented in Fig. 8(b). Mice treated with ZW760-mbc94 showed an ex vivo T/N ratio of 2.88 ($2.88\pm 1.20$), whereas blocked mice showed a T/N ratio of 1.54 ($1.54\pm 0.44$), with 47% blocking effect. The free dye control mice showed a T/N ratio of 1.28 ($1.28\pm 0.09$).

Fig. 8

Ex vivo optical imaging and biodistribution study. (a) Ex vivo imaging of tumor and selected organs 72-h PI of ZW760-mbc94, $\mathrm{ZW}760\text{-}\mathrm{mbc}94+4\mathrm{Q}3\mathrm{C}$, or free ZW760 dye. (b) Graphical representation of target/normal contrast ratio for ex vivo study, among three groups.

High-signal contrast in the liver was observed in both probe group ($\text{liver}/\mathrm{N}=24.54\pm 5.56$) and blocking group ($\text{liver}/\mathrm{N}=24.76\pm 1.44$), whereas the signal contrast in the kidney was much lower ($\text{kidney}/\mathrm{N}=2.69\pm 0.14$ in the probe group; $\text{kidney}/\mathrm{N}=2.68\pm 0.29$ in the blocking group). These results indicate that ZW760-mbc94 was cleared mainly from liver. The high-organ uptake caused relatively low in vivo tumor imaging contrast [Fig. 7(a)], as compared with the in vivo imaging results [Fig. 7(d) and Ref. 26] using our previously reported probe, NIR760-mbc94. Importantly, despite the high-probe uptake in the liver and kidney, blocking effect was only observed in the tumor, indicating specificity of ZW760-mbc94 in the living system.

4.

Summary

In summary, we developed a ${\mathrm{CB}}_2\mathrm{R}$-targeted zwitterionic NIR fluorescent probe, ZW760-mbc94, which showed specific binding to the target receptor in vitro and in vivo. When compared with the charged NIR probe (NIR760-mbc94) that possesses the same targeting moiety, ZW760-mbc94 showed enhanced blocking effect in vitro (from 40% to 50%) and ex vivo (from 35% to 47%). Overall, ZW760-mbc94 appears to be a promising ${\mathrm{CB}}_2\mathrm{R}$-targeted imaging probe. Moreover, replacing a charged NIR dye with a zwitterionic one appears to be an effective approach to reduce nonspecific binding.