Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

Abstract

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with (64)Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k(cat) and K(M) of 0.55+/-0.01 s(-1) and 1.12+/-0.06 microM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled (64)Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Fig. 1

Structure of caspase-3 activatable optical-nuclear molecular probe (LS498)

Fig. 2

Nonlinear fit of initial velocity with respect to substrate concentration and the corresponding Lineweaver-Burk plot (insert). Substrate concentrations varied from 270 nM to 35 μM.

Fig. 3

Imaging of multifunctional molecular probe in mice. (A) In vivo distribution of LS498 (Right) and non-quenched control analogue (Left) in mice at 30 minutes after intravenous injection of the imaging agents. Fluorescence of the activatable probe was efficiently quenched by greater than 16-fold relative to the non-quenched control. (B) Multimodal fluorescence (Left) and scintigraphic (Right) imaging of a mouse with subcutaneously implanted tubes containing ⁶⁴Cu-LS498 with BSA (left side) or caspase-3 (right side) 2 h after implantation. Tubes were implanted subcutaneously (about 1 mm) below the surface of the skin. Radioactivity is always “on” but fluorescence enhancement depends on the presence of caspase-3. The ratio of fluorescence intensity for the caspase-3 sample was 4.2 times greater than the control sample and this ratio increased to 5.6-fold after normalizing to radioactivity.