Selective Recognition of Bacteria Utilizing Zinc(II)-Dipicolylamine Conjugated Far-Red Fluorescent Probes

This dissertation evaluates a new family of near-infrared fluorescent molecular probes called squaraine rotaxanes for utility in whole animal imaging studies. Conjugating squaraine rotaxanes to zinc(II)-dipicolylamine (Zn-DPA) ligands produces probes that bind anionic phospholipids and related phosphate containing structures in the cell wall of bacteria. Planar fluorescence optical imaging proves that these Zn-DPA probes bind leg infections of Gram-positive 'Staphylococcus aureus' or Gram-negative 'Salmonella typhimurium' in living mice. High probe signal was visualized in the infected leg compared to the uninfected control leg. Control experiments showed that the Zn-DPA probes have little affinity for an inflammation response after intramuscular injection of λ-carrageenan into the mouse leg.

To quantify the affinity of Zn-DPA conjugated fluorophores for anionic bacterial membranes, a Zn-DPA with a conjugated far-red cyanine dye was employed in a Fšrster Resonance Energy Transfer assay using liposomes that mimic bacterial membranes. The binding constant to phosphatidylglycerol, a major component Gram-positive bacteria membranes, was determined to be 1.3 x 10^4 M-1 and the binding constant to lipid A, a major component of a Gram-negative bacteria, was determined to be 1.2 x 10^5 M-1. The Zn-DPA conjugated cyanine dye also bound bacterial cells in vitro and successfully targeted Gram-negative and Gram-positive infections upon intravenous treatment in living mice.

Another significant use of squaraine rotaxanes is shown through the development of squaraine rotaxane endoperoxides (SREP) as chemiluminescent-fluorescent imaging agents. The endoperoxide units within the squaraine rotaxane structure undergo a chemical reaction that produces near-infrared light. By encapsulating these self-illuminating SREPs within microparticle systems, it is possible to perform planar optical imaging at relatively deep tissue depths in living mice and rats. In addition, the delivery of microparticles to the lungs can be monitored via whole animal optical imaging. Although still in early stages, SREPs have tremendous potential for deep tissue imaging applications of various disease states in small animals.

Finally, a lipophilic fluorescent squaraine rotaxane encapsulated inside a lipid micelle, is shown to act as a targeting agent for brown fat in mice. By treating animals with as little as 1 nmol of dye, interscapular brown fat can be visualized by planar fluorescence imaging. In addition, accumulation of the lipophilic squaraine rotaxane in brown fat is shown to be significantly greater than a previously described fluorescent probe. Potential as a diagnostic tool is further supported by the colocalization of lipophilic squaraine rotaxane with 18FDG, a common brown fat imaging reagent used in positron emission tomography. Overall, the results in this dissertation indicate that squaraine rotaxanes are a promising molecular probe for optical molecular imaging.