Organocatalytic ring opening polymerization to access functional nanomaterials for the delivery of therapeutically relevant molecules and gene therapy targets
- The design and development of new materials that enable the uptake of drug targets across biological barriers has the potential to fundamentally transform modern drug discovery. A vast number of drug candidates are either ignored completely, or abandoned during development simply because they do not possess the proper physical properties needed to reach their biological targets. Here, we detail the development and use of new organocatalytic ring-opening polymerization methods to synthesize materials that can enable passage of therapeutically-relevant small molecules and biologics, previously thought of as undruggable, across in vitro and in vivo barriers. This work is directed at addressing that goal in three separate but interrelated areas: 1) Designing and synthesizing new functionalized monomers and polymeric materials with ideal drug delivery characteristics. 2) Mapping the molecular requirements for the delivery of therapeutic and diagnostic molecules with a variety of chemical and physical properties and challenges. 3) Using these transporters in therapeutic applications previously inaccessible without them. These three goals are manifest in several different areas, first in the design and evaluation of cell-penetrating peptide mimics for the delivery of small molecule drugs, followed by the synthesis of new, dynamic, polycationic materials for oligonucleotide and other polyanion delivery. First, we explore the critical need for delivery methods to expand our approaches for the treatment of disease, and the ways in which polymeric materials have been used to address this goal. Chapter 1 serves as an introduction to the goals addressed in this work, and more generally in the field of drug delivery. We then report the design, synthesis, and biological evaluation of a new family of highly-effective cell-penetrating molecular transporters based on an oligphosphoester backbone. These versatile delivery vehicles are easily accessed in two steps irrespective of oligomer length by the organocatalytic ring-opening polymerization (OROP) of 5-membered cyclic phospholane monomers. Collectively this study introduces a new and highly effective class of guanidinium-rich cell-penetrating transporters and methodology for their single-step conjugation to drugs and probes, and demonstrates that the resulting drug/probe-conjugates readily enter cells, outperforming previously reported guanidinium-rich oligocarbonates and peptide transporters. The remainder of this work is primarily focused on the complexation and delivery of oligonucleotides for gene therapy, specifically messenger RNA (mRNA). In the following 4 chapters, we highlight the impressive potential that mRNA delivery has for both the treatment of disease and advancement of academic research. The delivery of mRNA is akin to providing cells with the recipe that they need to produce their own drugs, which has astounding impacts in protein replacement therapy, vaccination, and genome editing. However, despite this potential, the delivery of these large, polyanionic molecules remains a substantial challenge. In Chapter 3, we describe the important aspects of this challenge and summarize the limitations of current approaches used for mRNA delivery. In Chapter 4, we move on to our preliminary work using amphipathic oligocarbonate transporters to complex and deliver mRNA to cultured cells, which we showed was unsuccessful due to the lack of an efficient mRNA release mechanism. We address this limitation in Chapter 5 by using a new class of dynamic biodegradable materials, charge-altering releasable transporters (CARTs). These oligo(carbonate-b--amino ester)s initially serve as polycations which electrostatically complex and deliver mRNA before rearranging to neutral small molecules to facilitate mRNA release. We highlight the synthesis and evaluation of these materials resulting in > 98% transfection in vitro and at high levels in vivo compared to commercially-available alternatives. In Chapter 6 we describe the synthesis of new CART materials that operate using a similar concept to the oligo(carbonate-b--amino ester)s but contain tunable functionality and rearrangement rates. This includes new formulation methods for the complexation of mRNA cargos with CART candidates to increase their stability and efficacy in vitro and especially in vivo. The work in Chapter 7 uses our CARTs in several therapeutically-relevant applications of mRNA delivery. In particular, we show that the in vivo delivery of antigen-encoding mRNA's to antigen-presenting cells can result in a robust immune response against tumors expressing that same antigen, resulting in protection from tumors in a prophylactic model, and tumor regression in an established tumor model. We have also shown that the delivery of Cas9 mRNA by CARTs can be used for efficient gene editing in vitro and in vivo. Also, in a combinatorial approach we demonstrate that combinations of CARTs containing different lipid portions can show preferential transfection of lymphocytes including T-cells and B-cells. In Chapter 8 we expand the use of CARTs to other oligonuceotdies including siRNA, pDNA, mcDNA, and miRNAs. Finally, in Chapter 9 we detail the application of the oligocarbonates and CARTs synthesized in the previous four chapters for the delivery of new polyanionic cargos. These cargos include inorganic polyphosphate (polyP), the inositol polyphosphates including diphospho-myo-inositol pentakisphosphate (5-InsP7), as well as photocaged analogs of ATP and the phospholipid diphosphatidylinositol-4,5-bisphosphate (PIP2).
|Type of resource
|electronic resource; remote; computer; online resource
|1 online resource.
|McKinlay, Colin James
|Waymouth, Robert M
|Wender, Paul A
|Waymouth, Robert M
|Wender, Paul A
|Xia, Yan, 1980-
|Degree committee member
|Xia, Yan, 1980-
|Stanford University, Department of Chemistry.
|Statement of responsibility
|Colin James McKinlay.
|Submitted to the Department of Chemistry.
|Thesis Ph.D. Stanford University 2018.
- © 2018 by Colin James McKinlay
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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