UC San Diego

The NEBI linkers were designed to undergo accelerated hydrolysis in mild aqueous acidic solutions, since a common mechanism for drugs to internalize into cells involves subcellular localization into the acidic endosomes (pH 5.5-6.0) or lysosomes (pH 4.5-5.0). The work described in this dissertation represents the first example of the use of pH-sensitive NEBI linkers for receptor-targeted drug delivery. Chapter 2 describes the synthesis of two novel indenoisoquinoline compounds, which are a class of TOP1 inhibitors that exhibit broad anti- cancer activity. These novel indenoisoquinolines contain a C-linked imidazole that can be incorporated directly into the NEBI linker. Targeted delivery of the indenoisoquinolines via folate will further increase their therapeutic efficiency. Chapters 3 and 4 describe the incorporation of the indenoisoquinolines in the development of several folate-conjugated DDSs that incorporate the NEBI linker. The in vitro cytotoxicity and imaging experiments are described in Chapter 5. Evaluation of the DDS in folate receptor (FR)-overexpressing and FR- knockdown cells suggest that cytotoxic activity is dependent on the presence of the folate group as well as on the acid-sensitive hydrolyzable group in the DDS. Microscopy studies demonstrate that, in FR-overexpressing cells, the DDS can internalize and localize in acidic lysosomal compartments, supporting a receptor-mediated mechanism for uptake and activation of the DDS. Finally, Chapter 6 describes the incorporation of the indenoisoquinolines conjugated to poly-lactic acid (PLA) nanoparticles via the acid-sensitive NEBI linker. This PLA -conjugated DDS was shown to be able to internalize into cells and has similar cytotoxic activity as the free active indenoisquinoline compound. This work further illustrates the versatility of the NEBI bifunctional linker