Advancement of Interpolyelectrolyte Complexes for the Delivery of Genetic Material

This dissertation focuses on the development and advancement of interpolyelectrolyte complexes (IPECs) and block ionomer complexes (BICs) for the delivery of genetic material, namely RNA, to cells, both human and insect. RNA interference (RNAi) provides a powerful tool for disease treatment and the elimination of crop pests at the genetic level. Therefore, development of successful delivery vehicles for its effector molecules, small interfering and double stranded RNAs (siRNA and dsRNA), is imperative. IPECs and BICs show the most promise as RNAi vectors, and thus this work focuses on ascertaining the structure-property relationships affecting RNA delivery as well as applying such insights toward enabling RNAi in crop pest insects that remain highly resistant to such treatment. In Section I, BIC-siRNA interactions and effectiveness in cell transfection are reported. Aqueous RAFT polymerization was used to prepare a series of hydrophilic-block-cationic copolymers in which the cationic block statistically incorporates increasing amounts of neutral, hydrophilic monomer such that the number of cationic groups remains unchanged but the cationic charge density is diluted along the polymer backbone. Reduced charge density decreases the electrostatic binding strength between copolymers and siRNA with the goal of improving siRNA release after targeted cellular delivery. However, lower binding strength resulted in decreased transfection and RNA interference pathway activation, leading to reduced gene knockdown. Enzymatic siRNA degradation studies with BICs indicated lowered binding strength increases susceptibility to RNases, which is the likely cause for poor gene knockdown. Section II discusses how RNAi-based technologies are ideal for pest control as they can provide species specificity and spare non-target organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal–Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) are efficiently taken up by cells, and can drive highly efficient gene knockdown. These IPECs also trigger target gene knockdown and moderate larval mortality when fed to fall armyworm larva. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices

Block Ionomer Complexes Consisting of siRNA and \u3ci\u3ea\u3c/i\u3eRAFT-Synthesized Hydrophilic-\u3ci\u3eBlock\u3c/i\u3e-Cationic Copolymers II: The Influence of Cationic Block Charge Density on Gene Suppression

Block ionomer complex (BIC)–siRNA interactions and effectiveness in cell transfection are reported. Aqueous RAFT polymerization was used to prepare a series of hydrophilic-block-cationic copolymers in which the cationic block statistically incorporates increasing amounts of neutral, hydrophilic monomer such that the number of cationic groups remains unchanged but the cationic charge density is diluted along the polymer backbone. Reduced charge density decreases the electrostatic binding strength between copolymers and siRNA with the goal of improving siRNA release after targeted cellular delivery. However, lower binding strength resulted in decreased transfection and RNA interference pathway activation, leading to reduced gene knockdown. Enzymatic siRNA degradation studies with BICs indicated lowered binding strength increases susceptibility to RNases, which is the likely cause for poor gene knockdown

Using Aldehyde Synergism to Direct the Design of Degradable Pro-Antimicrobial Networks

We describe the design and synthesis of degradable, dual-release, pro-antimicrobial poly(thioetheracetal) networks derived from synergistic pairs of aromatic terpene aldehydes. Initially, we identified pairs of aromatic terpene aldehydes derivatives exhibiting synergistic antimicrobial activity against Pseudomonas aeruginosa by determining fractional inhibitory concentrations. Synergistic aldehydes were converted into dialkene acetal monomers and copolymerized at various ratios with a multifunctional thiol via thiol-ene photopolymerization. The step-growth nature of the thiol-ene polymerization ensures every crosslink junction contains a degradable acetal linkage enabling a fully crosslinked polymer network to revert into its small molecule constituents upon hydrolysis, releasing the synergistic aldehydes as active antimicrobial compounds. A three-pronged approach was used to characterize the poly(thioether acetal) materials: (i) determination of the degradation/aldehyde release behavior, (ii) evaluation of the antimicrobial activity, and (iii) identification of the cellular pathways impacted by the aldehydes on a library of mutated bacteria. From this approach, a polymer network derived from a 40:60 p-bromobenzaldehyde:p-anisaldehyde monomer ratio exhibited potent antimicrobial action against Pseudomonas aeruginosa– a common opportunistic human pathogen. From a transposon mutagenesis assay, weshowed that these aldehydes target porins and multidrug efflux pumps. The aldehydes released from the poly(thioether acetal) networks exhibited negligible toxicity to mammalian tissue culture cells, supporting the potential development of these materials as dual-release antimicrobial biomaterial platforms

Ensuring operational compliance and ethical responsibility in the regulation of ART: the HFEA, past, present, and future

Under the Public Bodies Bill 2010, the HFEA, cornerstone in the regulation of assisted reproduction technologies (ART) for the last twenty years, is due to be abolished. This implies that there is no longer a need for a dedicated regulator for ART and that the existing roles of the Authority as both operational compliance monitor, and instance of ethical evaluation, may be absorbed by existing healthcare regulators. This article presents a timely analysis of these disparate functions of the HFEA, charting reforms adopted in 2008 and assessing the impact of the current proposals. Taking assisted conception treatment as the focus activity, it will be shown that the last few years have seen a concentration on the HFEA as a technical regulator based upon the principles of Better Regulation, with little analysis of how the ethical responsibility of the Authority fits into this framework. The current proposal to abolish the HFEA continues to fail to address this crucial question. Notwithstanding the fact that the scope of the Authority's ethical role may be questioned, its abolition requires that the Government consider what alternatives exists - or need to be put in place - to provide both responsive operational regulation and a forum for ethical reflection and decision-making in an area which continues to pose regulatory challenge

Mechanotransduction Pathways Linking the Extracellular Matrix to the Nucleus

Cells contain several mechanosensing components that transduce mechanical signals into biochemical cascades. During cell–ECM adhesion, a complex network of molecules mechanically couples the extracellular matrix (ECM), cytoskeleton, and nucleoskeleton. The network comprises transmembrane receptor proteins and focal adhesions, which link the ECM and cytoskeleton. Additionally, recently identified protein complexes extend this linkage to the nucleus by linking the cytoskeleton and the nucleoskeleton. Despite numerous studies in this field, due to the complexity of this network, our knowledge of the mechanisms of cell–ECM adhesion at the molecular level remains remarkably incomplete. Herein, we present a review of the structures of key molecules involved in cell-ECM adhesion, along with an evaluation of their predicted roles in mechanical sensing. Additionally, specific binding events prompted by force-induced conformational changes of each molecule are discussed. Finally, we propose a model for the biomechanical events prominent in cell–ECM adhesion