Design, Synthesis and Evaluation of Caged Melanin Precursors for Photoacoustic Imaging

Abstract

Photoacoustic imaging (PAI) is a relatively new and rapidly growing molecular imaging modality. Currently, this imaging modality is being used in both preclinical and clinical imaging applications. To date, significant efforts have been focused on developing exogenous PAI probes including various small molecule dyes and nanostructures. However, most of the preclinical agents’ clinical translation as potential PAI contrast agents is still limited due to their potential long-term toxicity. Melanin is an excellent tissue chromophore that can generate a stronger PA signal than any other tissue components such as hemoglobin. To date, a variety of melanin-based exogenous PA probes have been reported. However, the low solubility of synthetic melanin polymers in biologically relevant solvents as well as the inherent limitations of the nanoparticle-based imaging agents in biomedical applications can limit the full potential of melanin as a PA probe. Herein, we developed a platform that can use caged-melanin precursors as PAI agents instead of melanin. Our PAI agents can be used to generate melanin in situ based on the available tumor biomarkers in the site of interest upon generation of the uncaged melanin precursors from its caged version. We used both tyrosine and L-DOPA, the known melanin precursors, to develop our PAI agents. To explore our proposed strategy, initially we conjugated a known cathepsin B peptide substrate to the amino moiety of tyrosine and L-DOPA and evaluated the abilities of these agents to detect cathepsin B activity, a known tumor biomarker. Based on these initial results, we modified our design of the agents to include acylated peptide substrates and evaluated the performance of these new agents for detecting the activity of cathepsin-B. Both series of agents were able to form a melanin-based polymer as we anticipated, with faster kinetics observed with L-DOPA-based agents. However, these agents suffer from lack of specificity for cathepsin B activity and tyrosinase could still catalyze the oxidation of the caged agents to form quinone that led to melanin instead of only uncaged agents. We then developed a different approach by caging the hydroxyphenyl moiety of tyrosine with an acylated peptide substrate which we also evaluated for detecting cathepsin B activity. This agent also formed a melanin-based polymer and showed absolute specificity for detecting cathepsin B activity. These results establish hydroxyphenyl-caged agents as the initial structure from which to evaluate future variations, and for future PAI studies. We also investigated the development of other variations of this imaging platform that expand the functionality and utility of our research approach. Herein, we developed caged melanin precursors that can be used to detect β-glucuronidase activity and hydrogen peroxide with photoacoustic imaging. We designed two new contrast agents, β-glc-O-caged tyrosine and β-glc-O-caged L-DOPA, that connect glucuronic acid with tyrosine or L-DOPA. These agents were designed to produce eumelanin from the combination of β-glucuronidase and tyrosinase activities. Faster reactions were observed with the L-DOPA-based agent. These results establish β-glc-O-caged agents as the initial structures from which to evaluate future variations, and for future PAI studies. Additionally, we designed and synthesized a potential PAI agent that can be used to detect hydrogen peroxide activity. The L-DOPA generation based upon the activity of hydrogen peroxide on this agent is known, and it will be further investigated for future PAI studies. The dependency of the available tyrosinase at the site of interest to generate melanin from its natural precursors is a key feature of our approach. However, tyrosinase is not a tumor biomarker and it is known to overexpress only in melanoma cancer cells. Therefore, we decided to use melanoma cancer cell lines for future investigations of our platform. Additionally, we have decided to further develop our Ac-N-caged agents by introducing boronate ester moieties to protect the hydroxyl and catechol functionalities of those agents. Upon the success of this novel approach, we can then implement it to detect other biomarkers with PAI.