LLP2A-biotin-streptavidin-Alexa Fluor 680


Leung K.

Publication Details



In vitro Rodents



Optical fluorescence imaging is increasingly being used to observe biological functions of specific targets (1, 2). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging in small animals.

Integrins are a family of cell-surface heterodimeric glycoproteins that mediate diverse biological events involving cell–cell and cell–matrix interactions (3). They consist of an α and a β subunit, and they are important for cell adhesion and signal transduction. The α4β1 integrin plays an important role in hematopoiesis of lymphocytes; on the other hand, the α4β1 integrin affects tumor growth, tumor invasiveness, and metastasis (4). The α4β1 integrin is strongly expressed on lymphoid tumor cells (5). LLP2A was identified as a peptidomimetic ligand to bind to α4β1 integrin on human Jurkat T-lymphoid leukemia cells using “one-bead one-compound” combinatorial libraries (6). LLP2A was conjugated with Alexa Fluor 680 (Alexa680) via biotin and streptavidin (SA) interaction to study in vivo biodistribution of the tracer in tumor-bearing mice. Alexa680 is a NIR fluorescent dye with an absorbance maximum at 684 nm and an emission maximum at 707 nm with a high extinction coefficient of 183,000 (mol/L)-1cm-1. LLP2A-biotin-SA-Alexa680 was found to have a high specific accumulation in α4β1-positve lymphoid tumor cells in nude mice.



LLP2A and LLP2A-biotin were obtained using solid-phase synthesis (6). LLP2A-biotin was coupled to Alexa680-SA by use of the strong interaction between biotin and SA to form LLP2A-SA-Alexa680.

In Vitro Studies: Testing in Cells and Tissues


LLP2A exhibited a 50% inhibition concentration of 2.0 ± 1.4 pM in a cell-based, α4β1-mediated adhesion assay using Jurkat cells with a linear peptide (25 amino acids) of fibronectin (6). LLP2A was stable in human plasma for up to 18 days at 37°C. Flow cytometry and fluorescent microscopy confirmed the binding of LLP2A-biotin to α4β1-positive cells (Jurkat and Molt-4 leukemia cells) and not to α4β1-negative cells (A549 lung cancer cells). The binding to Molt-4 cells was inhibited by either LLP2A or anti-α4 monoclonal antibody HP2/1. Furthermore, LLP2A-biotin did not bind to cell lines transfected with α2β1, α5β1, α6β1, α9β1 (closely related to α4β1), αLβ2, or αMβ1, which indicated that LLP2A is specific to α4β1. LLP2A-biotin exhibited strong binding to Jurkat leukemia cells but weak binding to normal lymphocytes.

Animal Studies



Peng et al. (6) performed biodistribution studies of LLP2A-SA-Alexa680 in nude mice bearing a Molt-4 subcutaneous xenograft. Images were obtained after injection of 60 nmol/kg of LLP2A-SA-Alexa680. Substantial contrasts were observed between the Molt-4 tumor and normal tissue from 6 h until 44 h with a maximal difference at 24 h. Pretreatment with anti-α4 monoclonal antibody HP2/1 reduced fluorescent uptake into the tumor. Negligible fluorescence uptake into the tumor was observed with injection of SA-Alexa680 or scrambled LLP2A-SA-Alexa680. Ex vivo imaging showed that most of the LLP2A-SA-Alexa680 fluorescent signal intensity was seen in the Molt-4 tumors and kidneys. Cellular fluorescence was also observed in some blood vessels within the Molt-4 tumors.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R33 CA86364, R33 CA99136, U19 CA113298


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