Molecular Imaging | Projects | Collaborations | Resources

Back to Project List

Cell Penetrating, Caspase Activatable Near-Infrared Fluorescent Peptides

Molecular imaging probes have become increasingly useful for in vivo identification of intracellular processes. One area of research interest involves the development of cell-penetrating, near-infrared fluorescent peptide probes designed to be optically silent through intramolecular fluorescence quenching. Upon a biological event such as protease cleavage, the fluorophore-quencher pair is disrupted, activating the fluorescent reporter element and allowing for detection of the biological event. Previously, we developed a cell-penetrating near-infrared fluorescence probe based on this activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating peptide (HIV1 Tat) conjugated to an effector caspase (caspase-3) recognition sequence (DEVD) flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Upon exposure to effector caspases, the recognition sequence is cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. Currently, we are optimizing a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV), for in vivo applications. This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed higher quenching efficiency between the fluorophore-quencher pair, and was less toxic to cells. Assays using recombinant caspase enzymes revealed that the probe was specific for effector caspases (caspase 3 > 7 > 6) and cell culture models demonstrated probe activation specific to apoptotisis in a variety of cell types. In a rat model of retinal neuronal excitotoxicity, intravitreal injection of N-methyl-D-aspartate (NMDA) induced apoptosis of retinal ganglion cells (RGCs). Eyecup and retinal flat-mount images of NMDA-pretreated animals injected intravitreally with KcapQ using a clinically applicable protocol showed specific and widely distributed cell-associated fluorescence signals compared to untreated control animals. Fluorescence microscopy images of vertical retinal sections from NMDA-pretreated animals confirmed that activated probe was predominantly localized to RGCs and colocalized with TUNEL labeling. Thus, KcapQ is an improved effector caspase-activatable NIRF probe for enhanced noninvasive analysis of apoptosis in whole cells and live animals.
Figure 1
Schematic representation of KcapQ undergoing Caspase-3 activation.
Figure 2
Colocalization of TUNEL staining and KcapQ to retinal ganglion cells (RGC) by confocal fluorescence microscopy of vertical retinal sections from NMDA-pretreated eyes. (A) Differential interference contrast image of retinal cell layers. (B) Fluorescence image showing KcapQ activation predominantly in large cell bodies in the RGC layer. (C) Fluorescence immunohistochemistry showing TUNEL staining, again predominantly in the RGC layer. (D) Merged KcapQ fluorescence and TUNEL staining image. Brackets (]) indicate the nerve fiber layer (NFL), RGC layer, and inner nuclear layer (INL); asterisks indicate areas of colocalization of KcapQ and TUNEL positive signals in the RGC layer. Scale bar = 100 μm.
Reference:	Maxwell D, Chang Q, Zhang X, Barnett EM, Piwnica-Worms D. An Improved Cell-Penetrating, Caspase-Activatable, Near-Infrared Fluorescent Peptide for Apoptosis Imaging. Bioconjug Chem 2009 20(4): 702-9.	PubMed Link

Back to Project List