In Vivo Optical Imaging of Cell Death Using Fluorescent Synthetic Coordination Complexes

Structurally novel fluorescent molecular imaging probes were evaluated for ability to identify tissue containing dead and dying cells using whole-animal epi-fluorescence imaging and histological analysis. The near-infrared probe, PSS-794, with an appended Zn2BDPA affinity ligand, was evaluated in both subcutaneous and spontaneous prostate and mammary tumors animal models. In vivo imaging showed that PSS-794 had selectively accumulated in all tumors and targeting ratios were twice that of two control fluorophores. Ex vivo biodistribution and histological analyses suggest that PSS-794 targeted the necrotic regions of the tumors, which is consistent with in vitro microscopy. Furthermore, PSS-794 detected an increase in tumor cell death due to successful anti-tumor treatment in animal models employing focal beam radiation and chemotherapy.

The kinetic and perfusion properties of PSS-794 were assessed in two additional models of a cell death: a thymus atrophy mouse model and acute tissue damage model model. Uptake of PSS-794 in the atrophic thymi was significantly higher than control fluorophore at all time points, and the highest amount of PSS-794 accumulation occurred at 42 h after treatment. Fluorescent histological analyses of excised thymus tissue confirmed that PSS-794 targeted the dead and dying cells within the thymus. The tissue damage study used whole animal fluorescence imaging to visualize PSS-794 uptake at the site of injury, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a mechanistic similar but larger, fluorescent protein conjugate, Annexin V, showed less intense uptake at the site of muscle damage.

A preclinical mouse model for traumatic brain injury was adapted to investigate a set of complementary fluorescent imaging probes. Using non-invasive whole-body fluorescence imaging, PSS-794 permitted visualization of the injury in a living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The non-targeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicated that at three days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at seven days the blood-brain-barrier was still three times more permeable than before cryoinjury.

Finally, multivalent Zn2BDPA were evaluated to determine the whether increasing the number of Zn2BDPA ligands enhances cell death imaging. Imaging probes consisting of a fluorescent squaraine rotaxane scaffold with either two or four Zn2BDPA ligands were developed and their targeting properties were assessed in etoposide-treatedcells. Fluorescence microscopy showed that increasing the number Zn2BDPA units enabled lower concentrations of probe to be used while retaining high selectivity for dead and dying cells. When tested in three animal models of cell death, the tetra Zn2BDPA probe produced higher TT ratios than the analogous bis Zn2BDPA probe and an untargeted control fluorophore. An in vivo cold block study using an excess of Zn2BDPA inhibited accumulation of both the tetra and bis Zn2BDPA probe at the site of cell death. Overall, the results in this dissertation indicate that synthetic fluorescent Zn2BDPA conjugates are outstanding molecular probes for optical imaging of cell death.