Background

[PubMed]

Indocyanine 7 (Cy7)–tetrameric arginine-glycine-aspartic acid peptide (Cy7 E{E[c(RGDyK)]₂}₂) is an integrin-targeted molecular imaging agent developed for near-infrared (NIR) fluorescence imaging of tumor vasculature and tumor angiogenesis (1). Cy7 has a peak excitation wavelength of 743 nm and a peak emission wavelength of 767 nm.

Cellular survival, invasion, and migration control embryonic development, angiogenesis, tumor metastasis, and other physiologic processes (2, 3). Among the molecules that regulate angiogenesis are integrins, which comprise a superfamily of cell adhesion proteins that form heterodimeric receptors for extracellular matrix (ECM) molecules (4, 5). These transmembrane glycoproteins consist of two noncovalently associated subunits, α and β (18 α- and 8 β-subunits in mammals), which are assembled into at least 24 α/β pairs. Several integrins, such as integrin α_vβ₃, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin α_vβ₃ receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin α_vβ₃ receptor is generally not found in normal tissue, but it is strongly expressed in vessels with increased angiogenesis, such as tumor vasculature. It is significantly upregulated in certain types of tumor cells and in almost all tumor vasculature. Molecular imaging probes carrying the RGD motif that binds to the integrin α_vβ₃ can be used to image tumor vasculature and evaluate angiogenic response to tumor therapy (6, 7).

Various RGD peptides in both linear and cyclic forms have been developed for in vivo binding to integrin α_vβ₃ (8). It has been hypothesized that cyclic RGD peptide may have a faster rate of receptor binding or a slower rate of dissociation from the integrin α_vβ₃ than linear single RGD peptides (9). Wu et al. (9) proposed to evaluated whether a multimeric RGD peptide with >2 repeating cyclic RGD units would further enhance the affinity of the receptor-ligand interactions through a polyvalency effect. They also suggested that the increase in molecular size might prolong circulation time and reduce tumor washout rate. Optical imaging is an imaging method that utilizes light photons emitted from bioluminescence and fluorescence probes (7). Depth penetration is one major limiting factor in in vivo optical imaging. Currently, in vivo optical imaging has wide applications in small animal imaging but only limited applications in large animal and human studies (10). NIR fluorescence imaging (light range, 650−900 nm) has the advantages of relatively higher tissue penetration and lower autofluorescence from nontarget tissue. NIR fluorescent dyes conjugated RGD peptides such as Cy5.5-c(RGDyK), Cy5.5-c(RGDfK), and Cyp-GRD have been shown to visualize subcutaneously implanted integrin α_vβ₃−positive tumors (11-14). Wu et al. (1) investigated the use of the NIR fluorescent dye Cy7−conjugated tetrameric RGD to detect tumor integrin expression. Cy7 was chosen because of its deeper tissue penetration, lower scattering effect, and lower background autofluorescence compared to Cy5.5. The polyvalency effect and suitable apparent size of Cy7-E{E[c(RGDyK)]₂}₂ appeared to make it a highly potent integrin α_vβ₃ probe.

Synthesis

[PubMed]

Wu et al. (1) reported the synthesis of Cy7-E{E[c(RGDyK)]₂}₂ by using the monomeric cyclic RGD as the basic building block. Briefly, the fully protected linear pentapeptide H-D-Tyr(OtBu)-Lys(Boc)-Arg(Pbf)-Gly-Asp(OtBu)-OH was cleaved from the resin without affecting the side-chain protecting groups (15). Head-to-tail cyclization was then carried out in N,N-dimethylformamide. All side-chain protecting groups were then removed by treatment with trifluoroacetic acid (TFA) at room temperature for 1 h. The resulting c(RGDyK) was purified by high-performance liquid chromatography (HPLC). In the preparation of the tetrameric E{E[c(RGDyK)]₂}₂, Boc-Glu(OSu)-OSu was first prepared by mixing Boc-protected glutamic acid in N,N-dimethylformamide with N-hydroxysuccinimide and dicyclohexylcarbodiimide. The mixture was stirred at room temperature for 10 h. Boc-Glu(OSu)-OSu was obtained after this mixture was filtered to remove the dicyclohexylurea byproduct. c(RGDyK) was added to Boc-Glu(OSu)-OSu in N,N-dimethylformamide, and the pH was adjusted to 8.5. The mixture was stirred at room temperature overnight, and the Boc group was removed by TFA treatment to produce the dimeric E[c(RGDyK)]₂ with a 95% yield. This procedure was repeated with E[c(RGDyK)]₂ to produce the tetrameric E{E[c(RGDyK)]₂}₂ with a 92% yield.

Cy7-E{E[c(RGDyK)]₂}₂ conjugate was synthesized by conjugation of monofunctional hydroxysuccinimide of Cy7 (Cy7-NHS) with the α-amino group of glutamine residue (1). In this reaction, E{E[c(RGDyK)]₂}₂ in sodium borate buffer (pH 8.5) was mixed with Cy7-NHS in water at 4ºC. The reaction vessel was allowed to warm up to room temperature. TFA was added after 2 h of reaction, and the preparation was purified by HPLC to yield Cy7-E{E[c(RGDyK)]₂}₂. The Cy7-E{E[c(RGDyK)]₂}₂ conjugate was identified by matrix-assisted laser desorption/ionization–time-of-flight mass spectroscopy (MALDI-TOF) with a calculated molecular mass of 3,431.5 kDa.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The spectral properties of unconjugated Cy7 were reported to be 747 nm absorption maxima, 774 nm emission maxima, 0.28 quantum yield, and 200,000 M−¹cm−¹ molar extinction coefficient (16, 17). The ultraviolet absorption and emission spectra of Cy7-E{E[c(RGDyK)]₂}₂ were similar to that of unconjugated Cy7 in aqueous solution (1). No significant change in quantum yield was observed.

In vitro receptor binding assays of Cy7-E{E[c(RGDyK)]₂}₂ with integrin-positive U87MG cells and ¹²⁵I-echistatin, an integrin-specific radioligand, gave an inhibition concentration value (IC₅₀) of 23.1 ± 2.5 nM (n = 3) (1). In comparison, the IC₅₀ values for the Cy7-c(RGDyK) monomer and the Cy7-E[c(RGDyK)]₂ dimer were 70.1 ± 5.3 nM and 28.2 ± 2.8 nM, respectively. Cy7 conjugation appeared to cause minimal disturbance to receptor affinity. In comparison, the IC₅₀ values of fluorobenzoyl-c(RGDyK) and fluorobenzoyl-E[c(RGDyK)]₂ were reported to be 6.7 ± 0.5 nM and 3.5 ± 0.3 nM, respectively (18). Similar results were observed with U87AMG cell-staining studies. Only a weak signal was detected when the cells were incubated with the unconjugated Cy7. The fluorescence intensity of the stained cells followed the order of Cy7-E{E[c(RGDyK)]₂}₂ > Cy7-E[c(RGDyK)]₂ > Cy7-c(RGDyK)_.

Serum stability studies of incubating Cy7-E{E[c(RGDyK)]₂}₂ at 37ºC in complete mouse serum indicated that the Cy7 conjugate remained stable for 24 h (1).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NCI R21 CA102123, NIBIB R21 EB001785, NCI SAIRP R24 CA93862, ICMIC P50 CA114747, NCI CCNE US4.

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