Supramolecular Enhancement of Cell Permeation and Intracellular Targeting

While the framework for cytosolic delivery has been well established, there is a need to develop new ways to enhance cell permeation and intracellular targeting of drugs and imaging agents. Theranostics, which combines these features into one system, needs new molecular strategies to overcome undesirable differences in biodistribution and selectivity that currently exist between distinct therapeutic and diagnostic agents.

This thesis investigates different supramolecular methods to deliver fluorescent payloads into the cell including the use of nanoparticle delivery systems and small molecule imaging agents. Specifically, Chapter 2 evaluates the supramolecular properties of a new family of water-soluble hyperbranched polytriazoles that have a unique unimolecular micelle structure that could be broadly useful as a nanocarrier for various applications in therapy, imaging, or a combination of the two. Chapter 3 describes fundamental studies of four liposome systems that explored different supramolecular strategies for targeted liposome delivery and fusion, liposome leakage, and chemically triggered membrane fusion. Much of this work employs Synthavidin technology, which is based on the supramolecular capture of deep-red fluorescent squaraine dyes by tetralactam macrocycles. Subsequent chapters focus on microscopy using small molecule fluorescent probes. In Chapter 4, an intracellular fluorescence competition assay is developed to assess the capability of inhibitor candidate molecules to engage the biomedically important enzyme histone deacetylase 6 (HDAC6) inside living cells. The assay uses fluorescence cell microscopy and flow cytometry to quantify cell staining by a HDAC-selective fluorescent probe. In Chapter 5, two structurally related deep-red fluorescent probes are evaluated for microscopic imaging. One of the probes is a targeted fluorescent molecule with appended Ni-nitrilotriacetate groups that provided affinity for histidine-tagged proteins. The other probe is equipped with polyarginine peptide that improve cellular uptake. Together, these projects represent a new approach to intracellular microscopy using targeted deep-red fluorescent probes.