Date of Degree

9-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Mandë Holford

Committee Members

Dixie Goss

Hiroshi Matsui

Peter Prevelige

Subject Categories

Amino Acids, Peptides, and Proteins | Biochemistry | Biomedical and Dental Materials | Biophysics | Biotechnology | Macromolecular Substances | Medicinal Chemistry and Pharmaceutics | Molecular Biology | Nanomedicine | Other Chemicals and Drugs | Therapeutics

Keywords

Virus-like particle, VLP, P22 bacteriophage, peptide delivery, drug delivery, ROMP, disassembly, bioorthogonal, expanded genetic code

Abstract

The potency and specificity of bioactive peptides have propelled these agents to the forefront of pharmacological research. However, delivery of peptides to their molecular target in cells is a major obstacle to their widespread application. A Trojan Horse strategy of packaging a bioactive peptide within a modified protein cage to protect it during transport, and releasing it at the target site, is a promising delivery method. Recent work has demonstrated that the viral capsid of the P22 bacteriophage can be loaded with an arbitrary, genetically-encoded peptide, and externally decorated with a cell-penetrating peptide, such as HIV-Tat, to translocate across in vitro and in vivo models of the blood-brain barrier (BBB). However, disassembly of loaded capsids at the target site remains a challenge. Here, P22-derived nanocontainer systems for controlled disassembly and cargo release under physiological conditions are constructed, characterized, and tested. In particular, controlled disassembly in response to two types of bioorthogonal reactions is investigated: ring-opening metathesis polymerization (ROMP), and bimolecular "click" conjugation. It is shown that treatment of functionalized P22-derived nanocontainers with a water-soluble ruthenium catalyst results in ROMP and concomitant release of a GFP reporter under physiological conditions. In addition, genetic code expansion via amber suppression is used to construct self-assembling P22-derived nanocontainers that incorporate bioorthogonal handles for inverse electron-demand Diels-Alder cycloaddition (iEDDA) and strain-promoted azide-alkyne cycloaddition (SPAAC) in a site-specific manner. Functionalized nanocontainers are then shown to undergo morphological changes when treated with the corresponding bioorthogonal partner under physiological conditions.

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