Near-infrared fluorescence imaging of CD13 receptor expression using a novel Cy5.5-labeled dimeric NGR peptide

Abstract

In this study, we synthesized a novel Cy5.5-labeled dimeric NGR peptide (Cy5.5-NGR2) via bioorthogonal click chemistry, and evaluated the utility of Cy5.5-NGR2 for near-infrared fluorescence imaging of CD13 receptor expression in vivo. The dimeric NGR peptide (NGR2) was conjugated with an alkyne-containing PEG unit followed by mixing with an azide-terminated Cy5.5 fluorophore (Cy5.5-N3) to afford Cy5.5-NGR2. The probe was subject to in vitro and in vivo evaluations. The bioorthogonal click chemistry provided a rapid conjugation of the alkyne-containing NGR2 with Cy5.5-N3 in a quantitative yield within 15 min. The laser confocal microscopy revealed that binding of Cy5.5-NGR2 to CD13 receptor is target-specific as demonstrated in CD13-positive HT-1080 cells, CD13-negative MCF-7 cells, and a blocking study in HT-1080 cells. For in vivo optical imaging, Cy5.5-NGR2 exhibited rapid HT-1080 tumor targeting at 0.5 h postinjection (pi), and highest tumor-to-background contrast at 2 h pi. The CD13-specific tumor accumulation of Cy5.5-NGR2 was accomplished by a blocking study with unlabeled NGR peptide in HT-1080 tumor bearing mice. The tumor-to-muscle ratio of Cy5.5-NGR2 at 2 h pi reached 2.65 ± 0.13 in the non-blocking group vs. 1.05 ± 0.06 in the blocking group. The results from ex vivo imaging were consistent with the in vivo findings. We concluded that Cy5.5-NGR2 constructed by bioorthogonal click chemistry is a promising molecular probe, not only allowing the NIR optical imaging of CD13 overexpressed tumors, but also having the potential to facilitate noninvasive monitoring of CD13-targeted tumor therapy.

A CD44-specific peptide, RP-1, exhibits capacities of assisting diagnosis and predicting prognosis of gastric cancer

“…First, tissue auto-fluorescence can exist in in vivo fluorescence imaging, which may jeopardize the validity of the result. Therefore, a near-infrared fluorescein with higher efficient tissue penetration should be considered in further researches [37]. Second, although RP-1 had a relatively high sensitivity and specificity, false-positivity was still possible.…”

Cyanine Conjugate‐Based Biomedical Imaging Probes

“…Take a Cy5.5-labeled dimeric NGR peptide (21) as an example, the Asn-Gly-Arg (NGR) motif was discovered to home in on tumor vasculature by specifically binding CD13 receptor. [36] Another three peptide warheads screened out by phage display were covalently linked with cyanine fluorophores via short peptide linker (22)(23)(24) to target uPAR or c-Met for glioblastoma or colorectal cancer imaging, respectively. [37] When structural information of the target is available at atomic resolution, structure-based virtual screening becomes a practical technology with low cost and unprecedented speed; and accumu-lated literatures have documented the successful application in drug discovery.…”

“…[46] A class of urea-based PSMA inhibitors, including 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) and lysine-urea-glutamate (KuE), were utilized to conjugate with multifarious cyanine scaffolds and linker structures for intraoperative imaging of prostate cancer. [47] OTL78 (35) and YC-27 (36) are two representative conjugates based on DUPA and KuE, respectively. Other inhibitors, including huprine and SeISA, were also reported to conjugate with cyanine fluorophores for targeted imaging of acetylcholinesterase in neuromuscular junction (37), [48] and histone deacetylase 6 in breast tumor xenograft (38).…”

Design of multivalent fluorescent dendritic probes for site‐specific labeling of biomolecules

“…Moreover, scanners in microarray systems have the capacity for signal detection of Cy5. Most examples in literature detail the monovalent conjugation of Cy5 probes using simple bifunctional linkers . Dendrimers functionalized with Cy5 substituents, either at periphery or at focal point, have also been reported on as monodisperse scaffolds, including popular PAMAM or 2,2‐bis(methylol)propionic acid (bis‐MPA) architecture among others.…”

“…Dendrimers functionalized with Cy5 substituents, either at periphery or at focal point, have also been reported on as monodisperse scaffolds, including popular PAMAM or 2,2‐bis(methylol)propionic acid (bis‐MPA) architecture among others. The bioconjugation of Cy5 via covalent linkages to the target molecule often involves click chemistries although 1,2‐addition reaction, or reaction with amines through N ‐hydroxysuccinimide (NHS) chemistry have also been described. Of note is the use of hydrazide or hydrazine monovalent Cy5 probes, since they react selectively with carbohydrate on the Fc region of antibodies, with a well‐defined stoichiometry of conjugation, without affecting the binding site of the biomolecule.…”