Library Synthesis, Screening, and Discovery of Modified Zinc(II)-Bis(dipicolylamine) Probe for Enhanced Molecular Imaging of Cell Death

Abstract

Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

Nanoparticle targeting of Gram-positive and Gram-negative bacteria for magnetic-based separations of bacterial pathogens

“…This demonstrates the specificity of the ZnDPA targeting moiety over simply positively charged nanoparticles in LB media. These results are consistent with the previous reports that ZnDPA displays a strong nanomolar range affinity to phosphatidylserine ligands even in high salinity buffers, whereas simple charge interactions can be screened due to ionic strength (Plaunt et al 2014). …”

Zinc(II)-Dipicolylamine Coordination Complexes as Targeting and Chemotherapeutic Agents for Leishmania major

“…In vitro activity and toxicity measurements revealed that compound 7 is quite active against intracellular amastigotes and considerably less toxic against murine macrophages ( Table 1). The high tolerance of the ZnDPA complex matches previous observations of no obvious acute murine toxicity (22,27). However, more in vivo studies are needed to fully evaluate host toxicity and to more accurately measure the therapeutic window.…”

“…Parasite activity screening and host cell toxicity. The antileishmanial activities of eight ZnDPA complexes were evaluated against L. major promastigotes (22). The inhibitory activity was determined using a CellTiter-Blue assay, which measures the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin) (see Fig.…”

“…The targeted fluorescent ZnDPA probe mSeek, nontargeted fluorescent Control Dye, and all ZnDPA complexes tested were synthesized as previously reported (22,33). Microscopy slides and coverslips were purchased from Thermo Scientific.…”

“…1). Fluorescent ZnDPA probes are effective imaging agents for dead and dying mammalian cells (which expose anionic phosphatidylserine) in a range of cell culture systems and small animal models of disease (19)(20)(21)(22)(23)(24)(25)(26). ZnDPA probes also target the anionic surfaces of bacteria, and they have been used for imaging infection in animal models (27)(28)(29)(30)(31)(32)(33).…”