Second-generation fluorescent molecular imaging probes were evaluated for their ability to target dead and dying cells. This was accomplished using epi-fluorescence microscopy, whole-body fluorescence imaging and histological analysis. Two complimentary approaches for enhanced targeting to the anionic membrane were evaluated: (1) the addition of functionality groups to a single Zn-BDPA scaffold for potential secondary interactions at the membrane surface, and (2) the addition of multiple zinc(II)-bis(dipicolylamine) (Zn-BDPA) target ligands to a core squaraine rotaxane deep-red-emitting fluorophore. Lead candidate optical probes from approach 1 were evaluated in a subcutaneous prostate tumor model. Biodistribution analysis showed that the lead modified Zn-BDPA candidate selectively accumulated to the necrotic tumor tissue over 3-fold greater than the unmodified Zn-BDPA probe and 7-fold greater than the control dye with no attached target ligand. Ex vivo analysis also showed that the Zn-BDPA probes had selectively labeled the tumor necrotic foci. Approach 2 showed that the tetravalent Zn-BDPA probe had greater selectivity for anionic vesicles than the bivalent Zn-BDPA probe. Both probes were evaluated in cell culture and selectively targeted dead and dying cells over healthy cells. Interestingly, the divalent probe entered into the dead and dying cells, whereas the tetravalent probe only stained the membrane periphery.
The multivalent scaffold was also applied to bone targeting capabilities. Rather than Zn-BDPA targeting ligands, iminodiacetate groups were appended to the central squaraine rotaxane core. Bivalent and tetravalent iminodiacetate probes were evaluated in slices of bone tissue and in living mice. In vitro tests show that binding to bone was done in a calcium-dependent manner. In vivo results revealed that the tetravalent probe had enhanced affinity for the mouse skeleton that the bivalent version and it colocalized with a commercially available bisphosphonate bone-targeting near-infrared probe.
Zn-BDPA molecules were further explored as non-covalent triggers for triggering leakage of contents from vesicles. Preliminary work had triggered leakage of a quenched dye in cuvette studies. As an extension to this work, the water-soluble prodrug 5-aminolevulinic acid (5-ALA) was triggered from vesicles. This was further evaluated with modified versions of the trigger in the presence of cells for eventual protoporphyrin IX (PpIX) production. The modified trigger successfully improved 5-ALA leakage and interestingly improved PpIX cellular production greater than free 5-ALA. This phenomenon was further tested and revealed over 3-fold increase in PpIX cellular production using tyrosine Zn-BDPA triggers. The elucidated mechanism is a nontoxic “popping” of the membrane for greater 5-ALA cellular diffusion.
The last section of this dissertation uses novel light-absorbing molecules in applications of photothermal therapy (PTT). These molecules, croconaine dyes, are designed to absorb 800 nm laser light and generate sufficient heat to kill cancerous cells. Multiple probes were tested for photothermal output and the slightly more lipophilic version was able to selectively destruct cells. This was evaluated in standard viability assays and in a live-dead fluorescence microscopy assay. The lead candidate probe was also evaluated for PTT in a living prostate tumor mouse model. Results showed the suppression in tumor growth compared to the control mice for laser-only conditions.
Overall, the results in this dissertation indicate that next-generation small fluorescent molecular probes can enhance targeting to dead and dying cells. Other applications of these molecules include noncovalent triggered release of drug. Lastly, a novel strong-absorbing small molecule can achieve selective laser destruction of cells and tumor repression, proving it is an outstanding molecular probe for PTT.