| The landscape of chemotherapeutics is limited by ineffective cellular delivery. Controlled and efficient cellular drug delivery involves several stages: first, accumulation of active drug; second, association with the cell surface; third, internalization into the cell; and fourth, translocation into the cytosol. This thesis aims to investigate these stages with small molecules, all focused around a ribonuclease model system.;CHAPTER 1 serves as an introduction to cellular drug delivery, the pancreatic-type ribonuclease superfamily, and using these proteins as models to study drug delivery.;To optimize the first stage of cellular drug delivery, CHAPTER 2 reports on the attachment of a timed-release ribonucleoside 3'-phosphate pro-moiety to a chemotherapeutic to control accumulation of active drug. The human pancreatic ribonuclease was shown to release 4-hydroxytamoxifen from uridine-3'-phosphate. Timed-release of a chemotherapeutic could modulate active drug plasma concentration for safer and more efficacious chemotherapeutic delivery.;To facilitate the second and third stages of cellular drug delivery, CHAPTER 3 reports on the use of pendant boronic acids to mediate protein cell-surface association and cellular internalization. Boronic acids readily form boronate esters with cell-surface saccharides. Boronic acids were attached to bovine pancreatic ribonuclease, endowing the ribonuclease with increased affinity for saccharides and cellular internalization.;To enhance the fourth stage of cellular drug delivery, CHAPTER 4 discusses the use of a poly(amidoamine) (PAMAM) dendrimer in trans to potentiate the cytotoxicity of bovine pancreatic ribonuclease variants, presumably by increasing their translocation into the cytosol. Dendrimer causes no thermal destabilization to wild-type bovine pancreatic ribonuclease and only modest inhibition of its activity. Although this strategy is unlikely to be clinically useful, it adds to the understanding of this delivery stage.;CHAPTER 5 discusses future directions for research with pancreatic-type ribonucleases. APPENDIX 1 discusses the identification of a small-molecule antagonist of the interaction between a bovine pancreatic ribonuclease variant and the human ribonuclease inhibitor protein. APPENDIX 2 discusses the creation of a ribonuclease zymogen activated by urokinase. Together, this thesis describes progress towards understanding and facilitating chemotherapeutic delivery through the use of small molecules. |
