Near-infrared Fluorescent Divalent RGD Ligand for Integrin α_vβ₃-targeted Optical Imaging

Abstract

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)]₂ (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α_vβ₃-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α_vβ₃ binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.

Among the family of 24 integrins, integrin α_vβ₃ has become an important target for cancer imaging and therapy. This is because the up-regulation of integrin α_vβ₃ in cancer and endothelial cells is implicated in the progression and metastasis of various tumors. Significant progress has been made in the discovery and development of integrin α_vβ₃-specific lactam-based cyclic RGD peptide analogs such as cilengitide and c(RGDfK) for cancer therapy, as well as targeted delivery of cancer imaging and therapeutic agents.^1–9 Nevertheless, it remains a great challenge to leverage such molecules into successful clinical applications due to the complexities of integrin signaling network in cancer cells, tumor microenvironments, and in vivo environments. For example, Reynolds et al. ¹⁰ reported that nanomolar concentrations of RGD-mimetic integrin α_vβ₃ and α_vβ₅ inhibitors can promote VEGF-mediated tumor angiogenesis and tumor growth in mice, which may seriously compromise the therapeutic efficacy of this class of compounds. Therefore, novel approaches for exploiting integrin α_vβ₃ targeting are still needed to improve cancer imaging, diagnosis, and therapy.

As one of the most versatile imaging modalities, optical imaging allows noninvasive, sensitive, and real time imaging of molecular interactions and related functions in cells, tissues, and in vivo systems. In particular, optical imaging serves as a powerful tool for bridging the wide gap between preclinical and clinical studies, facilitating the discovery of optimal integrin α_vβ₃-targeted molecules, and their subsequent translational studies. ^11–14

We previously reported a series of carbocyanine-based near-infrared (NIR) fluorescent probes for tumor-targeted optical imaging in vivo. ^{12, 13, 15–21} Noteworthy, a dicarboxylic acid-containing carbocyanine analog (cypate; Figure 1) can be used for facile conjugations of various bioactive molecules on solid support and in solution for molecular targeting of proteins expressed on plasma membrane, including integrin α_vβ₃ and somatostatin receptors. Cypate has also served as a robust scaffold for conveniently constructing novel divalent and multivalent fluorescent molecules for tumor-targeted optical imaging. In the present work, we aimed to synthesize and evaluate a novel integrin α_vβ₃-targeted divalent RGD ligand based on cypate for potential use in optical imaging of tumors. We hypothesized that 1 can simultaneously bind two adjacent α_vβ₃ integrins to enhance the α_vβ₃-binding affinity and tumor-targeting with improved imaging contrast compared with its monovalent counterpart 2.

Figure 1.

Near-infrared fluorescent cypate and its RGD conjugates.

Initially, we prepared RGD peptide-dye conjugates from cypate and the non-protected cyclic peptide c(RGDfK) or c(RGDyK) via the reaction between two N-hydroxysuccinimidyl (NHS) esters of cypate and the amine group at the side chain of lysine residue as reported by others. ^{22, 23} However, we observed that the transformations of two carboxylic acid groups into the corresponding NHS esters were not complete despite using excessive amount of the coupling reagent DIC or EDCI. The additional EDCI could lead to activation of the carboxylic acid group at the side chain of Asp residue and related side reactions such as acylation of the free guanidine group in the Arg residues.²⁴ In a previous report, we demonstrated the successful synthesis of cypate-c(RGDfK) (2) by conjugating cypate with a protected cyclic RGD peptide, c[R(Pbf)GD(OBut)fK] 3. Consequently, we chose to use the same strategy for the synthesis of the divalent compound 1 in this study.¹⁹ The linear protected peptide H-D(OBut)-f-K(Dde)-R(Pbf)-G-OH was first assembled from H-Gly-2-chlorotrityl resin using the conventional Fmoc chemistry, followed by cleavage of the peptide from the resin with 1% TFA in DCM. The crude product was cyclized in the presence of PyBOP/HOBT/DIEA in dilute solution. The resulting cyclic protected peptide, c[R(Pbf)GD(OBut)fK(Dde)], was treated with 2% hydrazine in methanol to remove the Dde protecting group to form 3. Cypate was synthesized from the benzoindo-acid and glutaconaldehyde via its pre-acetylation intermediate in a good yield as reported previously. ^{16, 18} Cypate (1 equiv) was conjugated with 3 (3 equiv) in the presence of EDCI (6 equiv) and HOBT (3 equiv) in DMF (Scheme 1). The crude product, cypate-{cyclo[R(Pbf)GD(OBut)fK(~)]}₂ (4), was de-protected with TFA and purified by semi-preparative reverse phase HPLC to afford the desired divalent RGD conjugate 1. The compound was identified by analytical HPLC and LC-MS. As expected, 1, 2, and cypate showed similar UV-vis absorption and fluorescence emission spectra in 20% DMSO (λ_max/abs and λ_max/em: 783 nm and 811 nm, respectively). ¹⁶

Scheme 1.

Synthesis of the divalent RGD-cypate conjugates 1.

The ligand-receptor binding is a complex process that can be affected by many factors, including the radioligand tracer, binding affinity, and concentration used as well as the receptor and new ligands tested. Most of the integrin α_vβ₃ binding assays have been based on competitive displacement against ¹²⁵I-labeled echistatin. Echistatin, an RGD-containing polypeptide isolated from viper venom, belongs to the disintegrin family of inhibitory proteins. It binds to integrin α_vβ₃ with high affinity, but competitive binding with linear and cyclic RGD peptides have been achieved. Previously, we determined the integrin α_vβ₃ binding affinities of some RGD compounds using ¹²⁵I-labeled echistatin tracer as reported by Kumar et al. ^{16, 25} We also used the integrin α_vβ₃-specific ligand c(RGDyK) labeled with ¹²⁵I at D-tyrosine (y) residue instead of ¹²⁵I-echistatin for the competitive integrin α_vβ₃ binding assay. ¹⁷ The advantages of ¹²⁵I-c[RGDyK] over ¹²⁵I-echistatin include facile labeling, good quality control, and cost effectiveness.

In the present study, the radioactivity of c(RGDyK) peptide bound to integrin α_vβ₃ was determined with a gamma counter. Nonspecific binding of the tracer ¹²⁵I-c(RGDyK) was determined to be 5 to 10% of the total binding. The 50% inhibitory concentrations (IC₅₀) and affinity constant (Ki) were calculated by nonlinear regression analysis using the GraphPad Prism 4 data fitting software. The three RGD peptides, 1, 2, and c(RGDyK), were found to competitively inhibit the binding of ¹²⁵I-c(RGDyK) to α_vβ₃ in a concentration-dependent mode (Table 1). Compound 1 showed about 15 times stronger binding affinity than 2, while both c(RGDyK) and 2 exhibited comparable IC₅₀ values. In contrast, a scrambled analog, cypate-c(RGKDf), did not show significant inhibition compared with the other RGD compounds under the same condition (data not shown). The results further demonstrate the feasibility of using ¹²⁵I-c(RGDyK) as a tracer for integrin α_vβ₃ binding assays. Importantly, our study suggests that ¹²⁵I-c(RGDyK) is a reliable tracer for competitive binding assays of new compounds containing RGD structural motif with similar binding features and comparable binding affinity. Furthermore, ¹²⁵I-c(RGDyK) may serve as a sensitive method for studying some integrin α_vβ₃ ligands that could not compete favorably against ¹²⁵I-echistatin. We will further study the generality of ¹²⁵I-c(RGDyK) and compare it with ¹²⁵I-echistatin for studying integrin α_vβ₃-binding in future work.

Table 1.

The integrin α_vβ₃ binding of 1 and 2 traced by ¹²⁵I-c(RGDyK).

Compound	IC50 (nM, Mean ± SD)	Ki (Mean ± SD)	R2
1	0.281 ± 0.009	0.043 ± 0.015	0.957
2	4.305 ± 0.056	0.663 ± 0.086	0.9874
c(RGDyK)	3.52 ± 0.354	2.27 ± 0.228	0.9957

NIR fluorescence microscopy was used to evaluate the compounds for integrin α_vβ₃-mediated cellular binding and uptake. This also allowed the imaging of 1, 2, and cypate in integrin α_vβ₃ positive A549 cells at different time points. The cell staining results are dependent on the incubation time (Figure 2). Compound 1 showed higher uptake than both 2 and cypate after 1 h of incubation. Nevertheless, their intracellular fluorescence reached comparable levels at 6 h, suggesting that the longer incubation time increased the non-specific uptake. These results also reflect the complex process of integrin α_vβ₃-mediated cellular internalization of the fluorescent divalent RGD compounds, which may be governed by multiple factors such as receptor binding, receptor expression level, and endocytosis, as well as the structures and related physicochemical properties of the tested compounds.

Figure 2.

Binding and internalization of 1, 2, and cypate in A549 cells at different time points.

We further evaluated the subcellular distribution of 1, 2, and cypate by co-localization with MitoTracker Orange CM-TMRos® for the mitochondria (magenta) and DRAQ5 for the nucleus (blue). As shown in Figure 3, both 1 and 2 appear to have localized partially in the nucleus and the mitochondria. These results are consistent with previous reports by others. ^{26, 27}

Figure 3.

Subcellular distribution of 1 and 2 at 16 h of incubation. Fluorescent probes were excited (at 488 nm, 633 nm and 755 nm) and two-dimensional confocal images acquired by scanning a field at 2 µs per pixel. Each image shows overlay of nuclear stain (blue), mitochondrial stain (green) and cypate (red). Arrows point to cypate fluorescence indicative of the presence of the compound in the marked region.

To investigate integrin α_vβ₃ targeting and tumor localization in vivo, both 1 and 2 (100 µL, 60 µM) were intravenously injected by tail vein into nude mice bearing A549 tumor xenografts (5–7 mm diameter). Figure 4A shows the results of NIR fluorescence imaging (excitation and emission at 780nm and 830nm, respectively) at 1 h, 4 h, and 24 h post-injection. Compound 1 showed tumor localization in nude mice with good contrast between the tumor and non-target tissues at 4 h post-injection. In contrast, 2 had high accumulation in the liver and kidneys with low tumor uptake. Fluorescence intensity measurement shows that 1 had higher tumor/muscle ratio at 4 h and 8 h than 2 (Figure 4B). After euthanizing the animals at 24 h post-injection, the ex vivo organ analysis showed the relatively high uptake of 1 in the tumor and the liver compared with the kidneys, heart, and other tissues (Figure 4C and 4D). The results are in good agreement with the in vivo noninvasive optical imaging results.

Figure 4.

Optical imaging of 1 and 2 in mice. (A) In vivo optical imaging of tumors (circled areas) at 1, 4, and 24 h post –injection; (B) time-dependent tumor uptake; (C) ex vivo biodistribution of fluorescence at 24 h post-injection; and (D) ex vivo fluorescence imaging of representative organs.

Divalent and multivalent approaches have been widely studied in molecular discovery for improving the selectivity, specificity, and potency of receptor binding as well as the related biological and pharmacological activities.^{16, 28–32} Clearly, increased local concentration and divalent or multivalent interaction can enhance the integrin α_vβ₃ binding affinity of RGD compounds. Various divalent and multivalent RGD peptides for integrin α_vβ₃-targeted imaging such as ^18>F-FPPRGD2 and some fluorescent labeled RGD compounds have also been reported for tumor targeted imaging, therapy, and drug delivery. ^{14, 16, 26, 32–37} Especially, it is important to have the suitable linkage between two RGD motifs that can span the two neighboring integrin α_vβ₃ sites for simultaneous binding. As shown above, 1 represents a new type of NIR fluorescent divalent ligands using cypate as both linker and probe. Unlike other flexible linkers, the cypate motif consists of ploymethines and benzoindoline rings, which might help to pre-organize the divalent RGD ligand for simultaneous binding of two adjacent α_vβ₃ integrins with decreased binding entropy and enhanced stability of the resulting ligand-receptor complex.

By comparing with 2 and cypate, the significant improvement of 1 in tumor localization suggests the enhanced integrin α_vβ₃-targeting for tumor optical imaging. The results also support our hypothesis that the divalent compound can simultaneously bind to two adjacent integrin α_vβ₃ proteins and exhibit synergistic effects on receptor- and tumor-targeting. Furthermore, the linker of a divalent ligand plays an important role in achieving significant synergistic effects. ^{4, 31} The 27-atom length between the two RGD motifs of 1 may be sufficiently long to span two adjacent α_vβ₃ for simultaneous binding, leading to improvement in the integrin α_vβ₃ binding affinity and related tumor targeting (Figure 5). Therefore, compound 1 could serve as a prototype for constructing and optimizing novel integrin α_vβ₃-targeted compounds for the early diagnosis and targeted therapy of tumors.

Figure 5.

The two RGD motifs linked by cypate for simultaneous binding of two α_vβ₃ integrins.

There was high accumulation of 1 in the liver (Figure 4), which suggests its clearance from circulation through the reticuloendothelial system (RES). The strong NIR fluorescence in the mice over 24 h post-injection of 1 demonstrates the in vivo stability of 1 and its possible metabolites. As reported previously, NIR fluorescent IRDye800-RGD conjugates showed rapid clearance via the kidneys, and low tumor fluorescence ensued at 4 h post injection. ³⁸ These differences illustrate the important roles of the dye motif and the related hydrophilicity in the dynamics of divalent molecular imaging probes. These results also provide an insight into further structural modification of the cypate motif to minimize non-specific binding so as to enhance integrin α_vβ₃ and tumor-selective targeting.

Noteworthy, the modular approach we have used for synthesis of 1 allows for convenient preparation of such divalent conjugates in solution and / or on solid support. A vast array of molecules with distinct chemical and biological characteristics can be obtained by varying the peptide, linker, and cypate to elucidate the structure-activity relationship and mechanism of molecular targeting. In addition to integrin α_vβ₃, various cancer cell surface receptors such as somatostatin, growth factor, and steroid receptors associated with cancer initiation and progression have been reported. Therefore, cypate can serve as a unique fluorescent linker for conveniently constructing diverse divalent ligands to explore ligand-receptor and receptor-receptor interactions by optical imaging.

In summary, we have synthesized and evaluated a novel fluorescent divalent c(RGDfK) analog 1 based on cypate for integrin α_vβ₃ targeted optical imaging of tumors. The promising in vitro and in vivo data suggest that fluorescent divalent ligands of this type serve as an important strategy for exploring receptor-targeted optical imaging and provide insight into a better understanding of ligand design principles, structural optimization, and mechanism of action for specific receptor-targeting.