Macromolecular therapeutics like proteins and RNAs have immense potential to impact human health. Relative to small molecules, their architecture is significantly more complex and structurally diverse, enabling high-affinity and high-specificity interactions in the crowded cell interior. However, macromolecules (or biologics) are also typically charged and hydrophilic, and thus cannot directly cross a lipid bilayer. This results in cellular uptake through active pathways such as endocytosis which result in significant degradation unless the macromolecule can escape an endosome. Endosomal escape has been a bottleneck to the clinical application of biologics for decades, spurring the development of a multitude of delivery vehicles and assays to optimize them.

Here, we begin by providing a detailed review of the current state of the protein and RNA delivery field. We discuss techniques to establish absolute quantitation of exogenously delivered macromolecules in discrete locations within the cell, as well as an overview of delivery modalities that promote cytosolic and nuclear delivery of a variety of biologics. Next, we present a thorough interrogation of the mechanisms governing delivery of a cell-permeant miniature protein designed by the Schepartz lab called ZF5.3. Using a highly quantitative technique called fluorescence correlation spectroscopy (FCS), we establish molecular design rules for optimal cargos that expand the therapeutic scope of ZF5.3 and provide insights into its endosomal escape pathway. These findings are followed by the development of a highly sensitive tool to track the intracellular trafficking of ZF5.3. We exploit the increasing acidity of the endocytic pathway to design an assay that uses fluorescence lifetime as a readout for the pH microenvironment of ZF5.3. We apply this assay toward tracking both endosomal maturation and cytosolic localization of ZF5.3 to gain insights into the relationship between pH and intracellular delivery. Finally, we apply FCS to the nuclear delivery of a high-impact biologic, the Cas9 ribonucleoprotein complex (RNP), to establish a quantitative relationship between nuclear concentration and gene editing outcomes in multiple mammalian cell lines. We conclude with an outlook on the future of macromolecular delivery and the challenges that lie ahead. Together, the projects presented in this dissertation will contribute to the growing understanding of intracellular delivery pathways and provide a framework to optimize and quantify delivery strategies for biologics