Project Summary/Abstract Targeting of Bioorthogonal Chemotherapeutic Nanozymes to Tumor-Associated Macrophages In our proposed research we will use bioorthogonal chemistry to target tumor-associated macrophages (TAMs) in breast cancer, using bioorthogonal chemistry to turn them into ‘drug factory’ platforms for generation of chemotherapeutics at the tumor site. We will use our ‘nanozyme’ platform to encapsulate and protect transition metal catalysts (TMCs) within the monolayer of gold nanoparticles (AuNPs). These nanozymes will be targeted the mannose receptor strongly upregulated in TAMs. Systemic delivery of these nanozymes is anticipated to provide effective localization to TAMs that are highly accessible in tumors. Subsequent administration of non- toxic prodrugs will then provide uncaging of the chemotherapeutic localized to the tumor site. In our proposed research we will optimize the activity of our nanozyme platform. We will then engineer the nanozymes for selective TAM uptake through ‘stealth’ zwitterionic coating and suitable targeting elements. The targeting and therapeutic efficacy of the nanozymes will be quantified in vitro using mono- and co-culture models, Optimized particles will then be downselected for in vivo evaluation. Our specific aims are: Aim 1: Fabrication of Bioorthogonal Nanozymes. Goal: Engineering of monolayer structure to provide highly active and stable nanozymes. We will fabricate nanozymes coated with a zwitterionic layer to minimize non-specific uptake and mannose to target TAMs. We will optimize catalyst loading and stability in serum, and determine catalyst reactivity with prodrugs. Aim 2: In Vitro Activity and Targeting Studies. Hypothesis: Targeted nanozymes will provide highly cell-selective activation of prodrugs. We will quantify the intracellular activity of nanozymes in cells through catalytic uncaging of prodyes and prodrugs. Targeting efficacy to TAMs will assessed versus unpolarized macrophages and other cells, and in vitro therapeutic efficacy determined using co-culture models, optimizing the system based on specificity, efficacy/therapeutic window, and timing. Aim 3: Targeting of Prodrug Activation In Vivo. Hypothesis: Targeted nanozymes will localize prodrug activation to tumor sites, providing highly effective tumor therapy. We will use systemic injection of mannose- targeted nanozymes to activate profluorophores and prodrugs at tumor sites using 4T1 orthotopic breast carcinoma models. Quantitative tumor and intratumoral nanozyme distributions will be obtained using inductively-coupled mass spectrometry, and efficacy quantified by tumor size and mouse health. The overall goal of this project is to perform therapeutic ‘jiu-jitsu’, using TAMs that normally protect tumors to provide launch points for highly localized therapeutic delivery to tumors. This bioorthogonal therapeutic strategy is expected to reduce off-target effects and increase therapeutic efficacy relative to current chemotherapeutic approaches.

Project Narrative: Significance: This research is focused on the development of a new strategy for activation of therapeutics at tumor sites. This approach presents the possibility of generating active drugs only at tumor sites, reducing the off-target effects and minimizing health consequences for chemotherapy.