An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging

Abstract

Apoptosis is required for normal cellular homeostasis, and deregulation of the apoptotic process is implicated in various diseases. Previously, we developed a cell-penetrating near-infrared fluorescence (NIRF) probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an effector recognition sequence (DEVD) that was flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Once exposed to effector caspases, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. Herein, we present biochemical analysis of a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV). This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed an unexpectedly higher quenching efficiency between the fluorophore-quencher pair, and was potentially less toxic to cells. Assays using recombinant caspase enzymes revealed that the probe was specific for effector caspases (caspase 3 > 7 > 6). Analysis of apoptosis in HeLa cells treated with doxorubicin showed probe activation specific to apoptotic cells. 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 represents an improved effector caspase-activatable NIRF probe for enhanced noninvasive analysis of apoptosis in whole cells and live animals.

Figure 1

Schematic of KcapQ activation following cleavage by effector caspases. The DEVD cleavable sequence is flanked by an absorber (QSY21) and fluorophore (Alexa Fluor 647) which silence the reporter through intramolecular quenching. The cell-penetrating peptide sequence enables transport into the cell interior. Upon activation by caspase, the fluorophore is released inside target cells. The putative structure of Alexa Fluor 647 is from (45).

Figure 2

Chemical and biochemical characterization of KcapQ. A) UV-vis spectra of KcapQ (blue line), QSY21 (dash line) and Alexa Fluor 647 (red line). Notice the increase in absorbance at 605 nm. B) Recombinant enzyme assays of KcapQ normalized to mg of caspase for each enzyme ( caspase 3; caspase 7; caspase 6; ● caspase 9; ○ D-KcapQ). Assays with effector caspases resulted in KcapQ activation, whereas assays with initiator caspases showed no proteolytic activity towards KcapQ.

Figure 3

Inhibition curves of effector caspases interrogated with KcapQ. Increasing concentrations of the reversible inhibitor, DEVD-CHO, were incubated with 50 U caspase 3 (A), or 1 U caspase 7 (B) before the addition of KcapQ (0.5 μM). EC_50(i) values were determined from analysis of cleavage rates (ΔFU min^-1) of KcapQ using nonlinear regression curve fitting. Data are presented as mean values ± SEM (n = 2-3 each).

Figure 4

Representative fluorescence images of KcapQ activation during apoptosis in live cells. A) Doxorubicin-treated (10 M; 6 h) HeLa cells incubated with KcapQ (0.5 μM). B) Untreated HeLa cells incubated with KcapQ (0.5 μM). C) Doxorubicin-treated HeLa cells incubated with control D-KcapQ (0.5 μM). D) Untreated HeLa cells incubated with control D-KcapQ (0.5 M). Scale = 50 μm.

Figure 5

MTS cytotoxicity assays of KcapQ. Increasing concentrations of KcapQ (solid circle) and TcapQ (open circle) were incubated with HeLa cells for 48 h before the addition of MTS for analysis of cell viable mass. Data are presented as mean values ± SEM (n = 3 each).

Figure 6

Fluorescence images of activated KcapQ in retinal ganglion cells in an in vivo model of induced neuronal apoptosis. A) Fresh eye cup images of animals pretreated with NMDA for 16 h and imaged 2 h later with KcapQ or non-cleavable-D-KcapQ. Top Left: NMDA-pretreated eye imaged with KcapQ. Top Right: PBS-pretreated eye imaged with KcapQ. Bottom Left: NMDA-pretreated eye imaged with D-KcapQ. Bottom Right: PBS-pretreated eye imaged with D-KcapQ. B) Corresponding retinal flat mounts of eye cup samples. Top Left: NMDA-pretreated eye imaged with KcapQ. Top Right: PBS-pretreated eye imaged with KcapQ. Bottom Left: NMDA-pretreated eye imaged with D-KcapQ. Bottom Right: PBS-pretreated eye imaged with D-KcapQ. Scale = 100 μm. O, optic disc.

Figure 7

Co-localization of TUNEL staining and KcapQ to RGCs 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 retinal ganglion cell (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 co-localization of KcapQ and TUNEL positive signals in the RGC layer. Scale = 100 μm.