IRDye 800CW RGD Optical Probe: Tripeptide that Binds to Integrin Receptors for Cancer Studies.

Integrins are cell surface heterodimeric glycoproteins involved in cell-to-cell and cell-to-matrix interactions[1]. The recognition motif, RGD (Arg-Gly-Asp), is a tripeptide sequence used to bind integrin receptors including α_vβ₃. Interest in using a labeled RGD peptide ligand for the study and/or monitoring of diseases related to the α_vβ₃ receptor over-expression is increasing. Several groups have labeled RGD successfully with fluorescent dyes for in vitro and in vivo imaging[2,3].

Characterization of IRDye 800CW RGD included in vitro and in vivo binding to a variety of tumor cell lines (i.e., U87, A431, PC3M-LN4, and 22Rv1). Results from U87 and 22Rv1 cells are shown in Image A. Specificity of the conjugate for the integrin receptor was confirmed by competition with either unlabeled RGD peptide or unlabeled RAD, non-specific peptide, in U87 cells (Image B). In addition, Images C and D confirm IRDye 800CW RGD maintains specificity to the target tissue in in vivo applications. Images of nude mice bearing two subcutaneously implanted tumors on the hips illustrate IRDye 800CW RGD localized to tumor tissue (Image C). A dose of unlabeled RGD (given intravenously via the tail vein) prior to IRDye 800CW RGD gave reduced signal in both tumors confirming specificity (Image D).

A B

Figure: A) The On-Cell Western cell-based assay was used to evaluate binding affinity of IRDye 800CW RGD to U87 (glioblastoma) and 22Rv1 (prostate carcinoma) tumor cell lines. B) Binding of the IRDye 800CW RGD peptide was blocked by unlabeled RGD but not by a non-specific RAD peptide in U87 tumor cells. Nude mice bearing subcutaneous tumors, U87 (left hip) and A431(right hip), were imaged 24 hours post intravenous injection of IRDye 800CW RGD (1 nmole; C) or IRDye 800CW RGD (1 nmole) following a pre-injection 1 hour prior to an injection of unlabeled RGD peptide (100 nmoles; D) Images were captured on the Pearl Imager; 800nm signal is presented in pseudo-color overlaid on a white light image of the mouse. The images are normalized to the same look-up-table.

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Advantages of BrightSite™ Optical Agents

High signal-to-noise ratio.
Low NIR autofluorescence from tissues, cells, biological materials, and drug compounds.
IRDye 800CW absorption/emission near 800 nm matches NIR absorption minima for bodily fluids and tissues, resulting in excellent tissue penetration.

Molar extinction coefficient characteristics of water, hemoglobin and oxygenated hemoglobin (400-1000 nm). IRDye^® infrared dyes have ideal excitation/emission wavelengths for in vivo imaging.

IRDye^® 800CW RGD Optical Probe is composed of the recognition motif, RGD (Arg-Gly-Asp), a tripeptide sequence used to bind integrin receptors including a_vB₃. This receptor class is involved in tumor growth, tumor invasiveness, metastasis, tumor induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis.