Molecular targeting with nerve-binding peptides and activatable cell penetrating peptides (ACPPs) enables site-specific delivery of imaging agents or therapeutics in vivo. Nerve binding peptides conjugated to fluorescent dyes improve intraoperative identification of nerves, which should reduce accidental transection, thus reducing patient morbidities, and enhance nerve repair in pre-existing injury, improving outcomes. To enhance in vivo nerve contrast and advance the potential clinical application of first generation nerve-highlighting peptides, such as NP41, identifying molecular binding targets, improving affinity, reducing off-target binding, and validating function in human nerve is needed. Laminins-421, -211, and collagen VI were identified as NP41's binding targets using a novel tool developed to improve the isolation of low affinity interacting molecules, termed Ligand-Proximal Photooxidation (ProxPhOx). This approach uses light activation of a singlet-oxygen generating (SOG) fluorophore conjugated to a ligand to create a highly localized area of oxidation that can be covalently tagged with biotin hydrazide for purification. A new NP41-derivatized peptide, NP713, had higher affinity, in vivo contrast, and showed higher labeling of human nerve sections than NP41. Beyond improving nerve peptides, we sought to more specifically target disease sites using ACPPs. ACPPs sensitive to matrix metalloproteinases (MMPs), particularly MMP-2/-9, have been shown to target tumors and metastases; however, improvement in the enzyme selectivity and targeting of other proteases have the potential to increase uptake and specificity of localization to these and other disease sites. Detailed characterization of the selectivity of various substrates revealed potential enzyme targets, that when coupled to their application in disease models may reveal new biological insight, as well as creating potential diagnostic and therapeutic agents. Finally, intracellular delivery of non-membrane permeable therapeutics represents a major bottleneck in molecular targeting. The development of a novel assay for cytoplasmic delivery enabled quantification of membrane translocation efficiency and may allow identification of more effective carriers. The cumulative research presented here advances the use of nerve-targeted fluorophores and of ACPP-targeted drugs and contrast agents in the clinic. These agents will allow surgeons to accomplish difficult operations more precisely and will help clinicians to better diagnose and treat many diseases.
|Advisor:||Tsien, Roger Y.|
|Commitee:||Dowdy, Steven, Gianneschi, Nathan, Howell, Stephen, Nguyen, Quyen T.|
|School:||University of California, San Diego|
|School Location:||United States -- California|
|Source:||DAI-B 76/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Pharmacology|
|Keywords:||Fluorescence-guided surgery, Molecular imaging, Nerve imaging, Peptides, Phage display, Targeted therapeutic delivery|
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