Date of Award
5-2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemical and Biomolecular Engineering
Committee Chair/Advisor
Jessica Larsen
Committee Member
Sarah Harcum
Committee Member
Catherine Majors
Committee Member
Christopher Chouinard
Abstract
Messenger RNA (mRNA) therapeutics have emerged as a transformative modality in modern medicine, enabling rapid protein expression without genomic integration. Despite these promises, the clinical impact of mRNA technologies remains constrained by delivery challenges, including instability, endosomal entrapment, limited targeting specificity, and lack of stimulus-responsive control. This dissertation investigates redox-responsive polyamine-RNA complex coacervates as chemically programmable, phase-separated materials for mRNA delivery and oxidative stress sensing. By integrating principles of multivalent electrostatics, covalent crosslinking chemistry, and stimulus-triggered phase behavior, this work establishes a framework for engineering coacervates with tunable structural stability and redox-dependent responsiveness.
Coacervates, formed through liquid-liquid phase separation of oppositely charged macromolecules, offer high encapsulation efficiency, protection from enzymatic degradation, and environmental tunability. The dissertation further contextualizes reactive oxygen species (ROS) as clinically relevant biochemical triggers in oncology, radiotherapy, and space radiation environments, establishing oxidative stress as a programmable signal for therapeutic activation. Emerging polymeric nanocarrier strategies are analyzed to position coacervates within the broader landscape of RNA and protein delivery technologies.
Experimentally, this work introduces a novel redox-responsive crosslinking strategy using disulfide-containing 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP)-modified spermine to regulate multivalent interactions within polyuridylic acid v (polyU)/spermine coacervates. Mass spectrometry quantification demonstrates tunable modification of spermine species, enabling controlled crosslink density. Coacervation studies reveal that covalent crosslinking enhances long-timescale structural stability while preserving redox-triggered destabilization under reducing conditions. These findings establish that effective multivalency, rather than simple charge density, governs coacervate cohesion and phase stability.
Building on this foundation, oxidative destabilization is characterized under high hydrogen peroxide exposure and ionizing radiation. Distinct mechanistic differences are observed between reductive and oxidative triggers. Under acute oxidative stress, polyethylene glycol (PEG) stabilization inhibits turbidity decay through steric shielding, whereas PEG-diamine preserves electrostatic interactions and maintains redox responsiveness. Ionizing radiation experiments further demonstrate that redox-responsive destabilization can occur under radiation-relevant conditions, supporting potential applications in radiotherapy and space health. Additionally, compatibility with microfluidically generated polymersomes validates integration of coacervates into higher-order nanoparticle architectures, enabling modular hybrid delivery systems.
Beyond therapeutic delivery, this dissertation explores application of redox-responsive coacervates as oxidative stress sensors in CHO cell biomanufacturing. Coacervates form reproducibly in complex culture media and exhibit measurable turbidity and fluorescence changes in response to extracellular hydrogen peroxide. PEG-diamine stabilization enhances colloidal stability while retaining oxidative responsiveness, and incorporation of cell-penetrating peptides enables cellular interaction. These findings vi introduce a phase-based sensing paradigm for monitoring oxidative stress in manufacturing environments.
Collectively, this work advances complex coacervates from descriptive biomimetic condensates toward chemically engineered, stimulus-aware materials. By embedding covalent redox chemistry into polyamine backbones, this dissertation demonstrates programmable control over multivalent interactions, structural stability, and environmentally triggered phase transitions. The resulting platform integrates polymer chemistry, phase separation physics, redox biology, radiation science, and biomanufacturing applications. Redox-responsive polyamine-RNA coacervates therefore represent a versatile and modular technology for next-generation RNA delivery and oxidative stress sensing, with implications for oncology, radiation medicine, space health, and industrial biotechnology.
Recommended Citation
Forenzo, Chloe, "Redox-Responsive Polyamine-RNA Coacervates: Design, Characterization, and Applications in RNA Sensing and Delivery" (2026). All Dissertations. 4242.
https://open.clemson.edu/all_dissertations/4242
Author ORCID Identifier
0009-0006-9255-0537